Production of recombinant glycosidases fused with Usp45 and SpaX to avoid the purification and immobilization stages
The elucidation of the physicochemical properties of glycosidases is essential for their subsequent technological application, which may include saccharide hydrolysis processes and oligosaccharide synthesis. As the application of cloning, purification and enzymatic immobilization methods can be time consuming and require a heavy financial investment, this study has validated the recombinant production of the set of Lacticaseibacillus rhamnosus fucosidases fused with Usp45 and SpaX anchored to the cell wall of Lacticaseibacillus cremoris subsp cremoris MG1363, with the aim of avoiding the purification and stabilization steps. The cell debris harboring the anchored AlfA, AlfB and AlfC fucosidases showed activity against p-nitrophenyl α-L-fucopyranoside of 6.11 ± 0.36, 5.81 ± 0.29 and 9.90 ± 0.58 U/mL, respectively, and exhibited better thermal stability at 50 °C than the same enzymes in their soluble state. Furthermore, the anchored AlfC fucosidase transfucosylated different acceptor sugars, achieving fucose equivalent concentrations of 0.94 ± 0.09 mg/mL, 4.11 ± 0.21 mg/mL, and 4.08 ± 0.15 mg/mL of fucosylgalatose, fucosylglucose and fucosylsucrose, respectively.
| Main Authors: | , , , , |
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| Other Authors: | |
| Format: | artículo biblioteca |
| Language: | English |
| Published: |
Elsevier
2024-04-04
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| Subjects: | Enzyme stabilization, Fucosidase, Immobilization, Recombinant enzyme purification, Transfucosylation, |
| Online Access: | http://hdl.handle.net/10261/353836 http://dx.doi.org/10.13039/501100004837 http://dx.doi.org/10.13039/501100011033 http://dx.doi.org/10.13039/501100003339 https://api.elsevier.com/content/abstract/scopus_id/85189555786 |
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| Summary: | The elucidation of the physicochemical properties of glycosidases is essential for their subsequent technological application, which may include saccharide hydrolysis processes and oligosaccharide synthesis. As the application of cloning, purification and enzymatic immobilization methods can be time consuming and require a heavy financial investment, this study has validated the recombinant production of the set of Lacticaseibacillus rhamnosus fucosidases fused with Usp45 and SpaX anchored to the cell wall of Lacticaseibacillus cremoris subsp cremoris MG1363, with the aim of avoiding the purification and stabilization steps. The cell debris harboring the anchored AlfA, AlfB and AlfC fucosidases showed activity against p-nitrophenyl α-L-fucopyranoside of 6.11 ± 0.36, 5.81 ± 0.29 and 9.90 ± 0.58 U/mL, respectively, and exhibited better thermal stability at 50 °C than the same enzymes in their soluble state. Furthermore, the anchored AlfC fucosidase transfucosylated different acceptor sugars, achieving fucose equivalent concentrations of 0.94 ± 0.09 mg/mL, 4.11 ± 0.21 mg/mL, and 4.08 ± 0.15 mg/mL of fucosylgalatose, fucosylglucose and fucosylsucrose, respectively. |
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