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Protease-catalyzed synthesis of α-poly-L-Lysine and amphiphilic poly(L-lysine-co-L-phenylalanine) in a neat non-toxic organic solvent

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Abstract

Subtilisin Carlsberg (alkaline protease from Bacillus licheniformis) catalyzes the syntheses of high molecular weights (ca. 20 KDa) cationic α-poly-L-lysine and amphiphilic poly(α-L-lysine-co-L-phenylalanine) in neat organic solvent. The synthesis is conducted in liquid 1,1,1,2-tetrafluoroethane solvent, which is a hydrophobic non-toxic gas that does not deplete the ozone layer and approved for pharmaceutical applications. Solubility of substrates and adequate protease activity in this system with low water environment limits the reaction of hydrolysis of the growing peptide chains. The pressurization of this organic compressed fluid to liquid has low-pressure requirements (25 bar, 40 ºC), and its complete evaporation at atmospheric pressure after completing the reaction ensures solvent-free residues in products. The resulting polypeptides present null cytotoxicity according to MTT and NR analyses, as well as Calcein/EthD-1 assay in human cells.

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All data generated or analysed during this study are included in this published article [and its supplementary information files].

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Acknowledgements

We would like to thank CONACyT-Mexico for Postgraduate (AEG) and Postdoctoral (CHV) scholarships. MG thanks UNAM-DGAPA for funding project PAPIIT IN200123 and CONACyT for SEP-Frontera CF-2023-G-3. Authors would like to thank ENMEX SA de CV for the kind gift of SC sample, and Marisela Gutiérrez at USAII-FQ for FTIR analysis.

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Authors and Affiliations

  1. Departamento de Alimentos y Biotecnología, Facultad de Química, UNAM, Ciudad de Mexico, México

    Ángel Espinoza-González, Carmen Hernández-Valencia, Carmina Montiel & Miquel Gimeno

  2. Departamento de Ingeniería Química, Facultad de Química, UNAM, Ciudad de Mexico, México

    Luis Cedeño-Caero

  3. Unidad de Ingeniería de Tejidos Terapia Celular y Medicina Regenerativa, Instituto Nacional de Rehabilitación “Luis Guillermo Ibarra Ibarra”, Ciudad de Mexico, México

    Roberto Sánchez-Sánchez

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  1. Ángel Espinoza-González
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  2. Carmen Hernández-Valencia
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  4. Roberto Sánchez-Sánchez
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  5. Carmina Montiel
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  6. Miquel Gimeno
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Correspondence to Miquel Gimeno.

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Espinoza-González, Á., Hernández-Valencia, C., Cedeño-Caero, L. et al. Protease-catalyzed synthesis of α-poly-L-Lysine and amphiphilic poly(L-lysine-co-L-phenylalanine) in a neat non-toxic organic solvent. Bioprocess Biosyst Eng 46, 515–522 (2023). https://doi.org/10.1007/s00449-022-02836-3

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