Directed Enzyme Evolution
We published this book with the occasion of the 25th anniversary of the discovery of directed evolution. With the Nobel prize in Chemistry 2018 awarded to Prof. Frances Arnold, the volume is a tribute to pioneers of this thrilling research field, while providing a comprehensive overview of current research and the state-of-the-art.
The outstanding panel of contributors in “Directed Enzyme Evolution: Advances and Applications” has ensured the compilation of some remarkable examples of enzymes improved by evolution in just a single volume, as well as providing an interesting collection of the cutting-edge approaches and methods currently available or soon to be added to the directed evolution tool-box.
List of authors: Frances Arnold, Stefan Lutz, Manfred Reetz, Spencer Whitney, Miguel Alcalde, Ulrich Schwaneberg, Huimin Zhao, Tuck Seng Wong, Jose M. Sanchez Ruiz, Victor Guallar.
2025
Beltran-Nogal, A., Mateljak, I., Gonzalez-Perez, D. and Alcalde, M. (2025). Engineering unspecific peroxygenases by structure-guided in vivo recombination of homologous protein blocks. In: Methods in Enzymology: Biocatalysis. 714. https://doi.org/10.1016/bs.mie.2025.01.008
Keser, M., Dolz, M., Viña-Gonzalez, J., Gonzalez-Perez, D. and Alcalde, M. (2025). Protein purification protocols for recombinant enzymes produced in Pichia pastoris. In: Methods in Molecular Biology: Pichia pastoris: Methods and Protocols. Glieder, A., Kovar, K. and Garcia-Ortega, X. In press.
Menes-Rubio, A., Fernadnez-Garcia, A., Monterrey, D.T., Gomez de Santos, P., Sanchez-Moreno, I., Sanz-Aparicio, J. and Alcalde, M. 2025. Characterization of recombinant unspecific peroxygenase from Candolleomyces aberdarensis through crystallographic and substrate selectivity studies. ChemCatChem. 0: e202402015. https://doi.org/10.1002/cctc.202402015
2024
Sanchez-Moreno, I., Fernandez-Garcia, A., Mateljak, I., Gomez de Santos, P., Hofrichter, M., Kellner, H., Sanz-Aparicio, J. and Alcalde, M. (2024). Structural insights and reaction profile of a new unspecific peroxygenase from Marasmius wettsteinii produced in a tandem-yeast expression system. ACS Chemical Biology. 19: 2240−2253. https://doi.org/10.1021/acschembio.4c00504.
Dolz, M., Monterrey, D.T., Quartinello, F., Gomez de Santos, P., Mateljak, I., Pellis, A., Guebitz, G., Viña-González, J. and Alcalde, M. (2024). Enzyme benchmarking with polyethylene furanoate soluble scaffolds for directed evolution of PEFases. ACS Omega. 9 (45), 45633-45640. https://doi.org/10.1021/acsomega.4c09053.
Wang, Y., Domestici, C., Teetz, N., Holtmann, D., Alcalde, M., Wang, M., Qi, W., Zhang, W. and Hollmann, F. (2024). A Chemoenzymatic Cascade to Access Chiral β-hydroxy Ketones. Molecular Catalysis. 569: 114515. https://doi.org/10.1016/j.mcat.2024.114515.
Struwe, H., Grimm, C., Beutel, S., Dräger, G., Alcalde, M., Kirschning, A. and Kara, S. (2024). Expanding the “terpenome”: Applications of unspecific peroxygenases (UPOs) in oxidations of unnatural terpenoids. ChemCatChem. 0: e202401414 doi.org/10.1002/cctc.202401414
2023
Dolz, M., Monterrey, D.T., Beltrán-Nogal, A., Menés-Rubio, A., Keser, M., González-Pérez, D., Gómez de Santos, P., Viña-González, J. and Alcalde, M. (2023) The Colors of Peroxygenase Activity: Colorimetric High-Throughput Screening Assays for Directed Evolution. In: Methods in Enzymology: P450 enzymes. Renata, H. Ed. Elsevier.Vol 693: 73-109. https://doi.org/10.1016/bs.mie.2023.09.006
Gomez de Santos, P., Mateljak, I., Hoang, M.D., Fleishmann, S.J., Hollmann, F. and Alcalde, M. (2023). A repertoire of computationally designed peroxygenases for enantiodivergent C-H oxyfuntionalization reactions. Journal of the American Chemical Society 145: 3443–3453. https://doi.org/10.1021/jacs.2c11118
Gomez de Santos, P., Gonzalez-Benjumea, A., Fernandez-Garcia, A., Aranda, C., Wu, Y., But, A., Molina-Espeja, P., Mate, D., Gonzalez-Perez, D., Zhang, W., Kiebist, J., Scheibner, K., Hofrichter, M., Swiderek, K., Moliner, V., Sanz-Aparicio, J., Hollmann, F., Gutierrez, A. and Alcalde M. (2023). Engineering a highly regioselective fungal peroxygenase for the synthesis of hydroxy fatty acids. Angewandte Chemie International Edition 62:e202217372. https://doi.org/10.1002/anie.202217372
Hilberath, T., van Oosten, R., Victoria, J., Brasselet, H., Alcalde, M., Woodley, J. and Hollmann, F. (2023). Towards kilogram-scale peroxygenase-catalyzed oxyfunctionalization of cyclohexane. Organic Process Research & Development 27: 1384-1389. https://doi.org/10.1021/acs.oprd.3c00135
Xu, X., Mao, Y., Martinez, M., Roda, S., Floor, M., Guallar, V., Paul, C.E., Alcalde, M. and Hollmann, F. (2023). Peroxygenase-catalysed selective oxidation of silanes to silanols. Angewandte Chemie International Edition e202302844. https://doi.org/10.1002/anie.202302844
De Santis, P., Wegstein, D., Burek, B.O., Patzsch, J., Alcalde, M., Kroutil, W., Blot, J.Z. and Kara, S. (2023). Robust light driven enzymatic oxyfunctionalization via immobilization of unspecific peroxygenase. ChemSusChem e202300613. https://doi.org/10.1002/cssc.202300613
Karich, A., Salzsieder, F., Kluge, M., Alcalde, M., Ullrich, R. and Hofrichter, M. (2023). Conversion of unsaturated short- to medium-chain fatty acids by unspecific peroxygenases (UPOs). Applied Microbiology 3: 826-840. https://doi.org/10.3390/applmicrobiol3030057
Wang, Y., Teetz, N., Holtmann, D., Alcalde, M., van Hengst, J.M.A., Liu, X., Wang, M., Qi, W., Zhang, W. and Hollmann, F. (2023). Selective peroxygenase-catalysed oxidation of toluene derivatives to benzaldehydes. ChemCatChem 15: e202300645. https://doi.org/10.1002/cctc.202300645
Meyer, L.E., Horvath, D., Vaupel, S., Meyer, J., Alcalde, M. and Kara, S. (2023). A 3D printable synthetic hydrogel as an immobilization matrix for continuous synthesis with fungal peroxygenases. Reaction Chemistry & Engineering 8: 984-988. https://doi.org/ 10.1039/d3re00058c
Wu, Y., Paul, C.E., Hilberath, T., Jongkind, E.P.J., Alcalde, M. and Hollmann, F. (2023). Peroxygenase-promoted enzymatic cascades for the valorisation of fatty acids. ChemCatChem e202300411. https://doi.org/10.1002/cctc.202300411
Domestici, C. Wu, Y., Hilberath, T., Alcalde, M., Hollmann, F. and Macchioni, A. (2023). Hybrid Organometallic and Enzymatic Tandem Catalysis for oxyfunctionalisation Reactions. ChemCatChem e202201623. https://doi.org/10.1002/cctc.202201623
Monterrey, D.T., Menés-Rubio, A., Keser, M., Gonzalez-Perez, D. and Alcalde, M. (2023). Unspecific peroxygenases: The pot of gold at the end of the oxyfunctionalization rainbow?. Current Opinion in Green and Sustainable Chemistry 41: 100786. https://doi.org/10.1016/j.cogsc.2023.100786
2022
Barber-Zucker, S., Mateljak,I., Goldsmith, M., Kupervaser, M., Alcalde, M. and Fleishman, S.J. (2022). Functional diversity in designed high-redox potential laccases. ACS Catalysis 12: 13164–1317. https://doi.org/10.1021/acscatal.2c03006
Gutierrez-Rus, L.I., Alcalde, M., Risso, V.A. and Sanchez-Ruiz, J.M. (2022). Efficient base-catalyzed Kemp elimination in na engineered ancestral enzyme. International Journal of Molecular Science 23: 8934. https://doi.org/10.1101/2022.07.28.501888
Sang, X., Tong, F., Zeng, Z, Wu, M., Yuan, B., Sun, Z., Sheng, X., Qu, G., Alcalde, M., Hollmann, F. and Zhang, W. (2022). Biocatalytic plaftorm for the synthesis of enantiopure propargylic alcohols and amines. Organic Letters 24: 4252–4257. https://doi.org/10.1021/acs.orglett.2c01547
Beltran-Nogal, A., Sanchez-Moreno, I., Mendez-Sanchez, D., Gomez de Santos, P., Hollmann, F. and Alcalde, M. (2022). Surfing the wave of oxyfunctionalization chemistry by engineering fungal unspecific peroxygenases. Current Opinion in Structural Biology 73: 102342. https://doi.org/10.1016/j.sbi.2022.102342
Barber-Zucker, S., Mindel, V., Garcia-Ruiz, E., Weinstein, J.J., Alcalde, M. and Fleishman, S.J. (2022). Stable and functionally diverse versatile peroxidases by computational design directly from sequence. Journal of the American Chemical Society 144: 3564-3571. https://doi.org/10.1021/jacs.1c12433
Dolz, M., Gomez-Fernandez, B., Sanchez-Moreno, I., Mendez-Sanchez, D., Viña-Gonzalez, J. and Alcalde, M. (2022). Colorimetric high-throughput screening assay to engineer fungal peroxygenases for the degradation of thermoset composite epoxy resins Frontiers in Catalysis 2: 883263. https://doi.org/10.3389/fctls.2022.883263
Hilberath, T., Alcalde, M. and Hollmann, F. (2022). Assessing peroxygenase-mediated oxidations in the presence of high concentrations of water-miscible co-solvents Frontiers in Catalysis. 2: 882992. https://doi.org/10.3389/fctls.2022.882992
Xu, X., Brasselet, H., Jongkind, E., Alcalde, M., Paul, C. and Hollmann, F. (2022). A peroxygenase-alcohol dehydrogenase cascade reaction to transform ethylbenzene derivatives into enantioenriched phenylethanols. ChemBioChem 23: e2022200017. http://doi.org/10.1002/cbic.202200017
2021
Gomez de Santos, P., Hoang, M.D., Kiebist, J., Kellner, H., Ullrich, R., Scheibner, K., Hofrichter, M., Liers, C. and Alcalde, M. (2021). Functional expression of two unusual acidic peroxygenases from Candolleomyces aberdarensis in yeasts by adopting evolved secretion mutations. Applied & Environmental Microbiology 87: e00878-21. https://doi.org/10.1128/AEM.00878-21
Molina-Espeja, P., Beltran, A., Alfuzzi, M.A., Guallar, V. and Alcalde, M. (2021). Mapping potential determinants of peroxidative activity in an evolved fungal peroxygenase from Agrocybe aegerita. Frontiers in Bioengineering and Biotechnology 9: 741282 https://doi.org/10.3389/fbioe.2021.741282
Mateljak, I. and Alcalde, M. (2021). Engineering a highly thermostable high-redox potential laccase. ACS Sustainable Chemistry and Engineering 9: 9632-9637. https://doi.org/10.1021/acssuschemeng.1c00622
Martin-Diaz, J., Molina-Espeja, M., Hofrichter, M., Hollman, F. and Alcalde, M. (2021). Directed evolution of the unspecific peroxygenase from Agrocybe aegerita in organic solvents. Biotechnology & Bioengineering 118: 3002-3014.https://doi.org/10.1002/bit.27810
Miresela, R., Ramirez-Ramirez, J., Alcalde, M. and Ayala, M. (2021). Ether oxidation by an evolved fungal peroxygenase: insights into substrate recognition and reactivity. Journal of Fungi 7: 608. https://doi.org/10.3390/jof7080608
Wilbersa, D., Brehm, J., Lewis, R.J., van Marwijk, J., Davies, T.E., Morgan, D.J., Opperman, D.J., Smit, M.S., Alcalde, M., Kotsiopoulos, A., Harrison, S.T.L., Hutching, G.J. and Freakley, S.J. (2021). Controlling product selectivity with nanoparticle composition in tandem chemo-biocatalytic styrene oxidation. Green Chemistry 23: 4170-4180. http://DOI: 10.1039/d0gc04320f
Friederike E. H. Nintzel, F.E.H., Yinqi Wu, Y., Matteo Planchestainer, M., Held, M., Alcalde, M. and Hollmann, F. (2021). An alginate-confined peroxygenase-CLEA for styrene epoxidation. Chemical Communications 57: 5766-5769.https://doi.org/10.1039/d1cc01868j
Tonin, F., Tieves, F., Willot, S., van Troost, A., van Oosten, R., Breestraat, S., van Pelt, S., Alcalde, M. and Hollmann, F. (2021). Pilot-scale production of the peroxygenase from Agrocybe aegerita. Organic Process Research & Development 25: 1414-1418. https://doi.org/10.1021/acs.oprd.1c00116
Püllmann, P., Knorrscheidt, A., Münch, J., Palme, P.R., Hoehenwarter, W., Marillonnet, S., Alcalde, M., Westermann, B. and Weissenborn, M.J. (2021). A modular two yeast species secretion system for the production and preparative application of fungal peroxygenases. Communications Biology 4: 562.https://doi.org/10.1038/s42003-021-02076-3
Viña-Gonzalez, J. and Alcalde, M. (2021). Directing the evolution of the fungal ligninolytic secretome. In: Protein Engineering: Tools and Applications. Zhao, H., Lee, S.Y., Nielsen, J. Stephanopoulos, G. Ed. Wiley-VCH, Weinheim, Germany. 295-311. ISBN: 978-3-527-34470-3.
Zhang, W., Li, H. Younes, S., Gómez de Santos, P., Tieves, F., Grogan, G., Pabst, M., Alcalde, M., Whitwood, A. and Hollmann, F. (2021). Biocatalytic aromaticity-breaking epoxidation of naphthalene and nucleophilic ring-opening reactions ACS Catalysis 11: 2644-2649. https://doi.org/10.1021/acscatal.0c05588
Hobisch, M., Holtmann, D., Gomez de Santos, P., Alcalde, M., Hollmann, F. and Kara, S. (2021). Recent developments in the use of peroxygenases – exploring their high potential in selective oxyfunctionalisations. Biotechnology Advances 51: 107615. https://doi.org/10.1016/j.biotechadv.2020.107615.
Yayci, A., Dirks, T., Kogelheide, F., Alcalde, M., Hollmann, F., Awakowicz, P. and Bandow, J. (2021). Protection strategies for biocatalytic proteins under plasma treatment. Journal of Physics D: Applied Physics 54: 035204. https://doi.org/10.1088/1361-6463/abb979
2020
Gomez de Santos, P., Lazaro, S., Viña-Gonzalez, J., Hoang, M.D., Glieder, A., Hollmann, F. and Alcalde, M. (2020). Evolved peroxygenase-aryl alcohol oxidase fusions for self-sufficient oxyfunctionalization reactions. ACS Catalysis 10: 13524-13534. https://dx.doi.org/10.1021/acscatal.0c03029
Wuyuan, Z., Liu, H., van Schie, M., Hagedoorn, P.L., Alcalde, M., Denkova, A., Djanashvili, K. and Hollmann, F. (2020). Nuclear Waste and Biocatalysis. A Sustainable Liaison?. ACS Catalysis. 10: 14195-14200. https://dx.doi.org/10.1021/acscatal.0c03059
Yayci, A., Dirks, T., Kogelheide, F., Alcalde, M, Hollmann, F., Awakowicz, P. and Bandow, J.E. (2020). Microscale atmospheric pressure plasma jet as a source for plasma-driven biocatalysis. ChemCatChem. 12: 5893-5897. doi.org/10.1002/cctc.202001225
Ramirez-Ramirez, J., Martin-Diaz, J. Pastor, N., Alcalde, M. and Ayala, M. (2020). Exploring the effect of the topography of the active site on the oxidative capacity of the unspecific peroxygenase variant PaDa-I. International Journal of Molecular Science 21: 5734. https://doi.org/10.3390/ijms21165734
Gomez-Fernandez, B., Risso, V.A., Rueda, A., Sanchez-Ruiz, J.M. and Alcalde, M. (2020). Ancestral resurrection and directed evolution of fungal Mesozoic laccases. Applied and Environmental Microbiology. 86:e00778-20. JOURNAL COVER & SPOTLIGHT. https://doi.org/10.1128/AEM.01732-20
Viña-Gonzalez, J. And Alcalde, M. (2020). In vivo site-directed recombination (SDR): An efficient tool to reveal beneficial epistasis. In: In: Enzyme engineering and evolution: general methods. Methods in Enzymology 643. Tawfik, D. Ed. Academic Press Elsevier Inc, Cambridge, MA, EEUU. Pages 1-12. ISBN 978-0-12-821149-6. https://doi.org/10.1016/bs.mie.2020.04.021.
Mateljak, I., Gomez-Fernandez, B. and Alcalde, M. (2020). Laccase engineering by directed and computational evolution. In: Laccase in bioremediation of pollutants and xenobiotics. Bioremediation and Waste valorization. Schlosser, D., Ed. Springer Nature Switzerland. Pages: 191-206. ISBN: 978-3-030-47906-0. https://doi.org/10.1007/978-3-030-47906-0_8.
Viña-Gonzalez and Alcalde, M. (2020). Directed evolution of the aryl-alcohol oxidase: beyond the lab bench. Computational and Structural Biotechnology Journal. 18: 1800-1810. https://doi.org/10.1016/j.csbj.2020.06.037
Yuan, B., Mahor, D., Fei, Q., Wever, R., Alcalde, M., Zhang, W. and Hollmann, F. (2020). A Water-Soluble Anthraquinone Photocatalyst Enables Methanol Driven Enzymatic Halogenation and Hydroxylation Reactions. ACS Catalysis. 10: 8277-8284. https://doi.org/10.1021/acscatal.0c01958
Gomez-Fernandez, B., Risso, V., Sanchez-Ruiz, J.M. and Alcalde, M. (2020). Consensus design of an evolved high-redox potential laccase. Frontiers in Bioengineering and Biotechnology. 8, 354: 1-11. doi: 10.3389/fbioe.2020.00354
Hobish, M., van Schie, M., Kim, J., Andersen, K.R., Alcalde, M., Kourist, R., Park, C.B., Hollmann, F. and Kara, S. (2020). Solvent-free photobiocatalytic hydroxylation of cyclohexane. ChemCatChem. 12: 4009-4013. JOURNAL COVER. https://doi.org/10.1002/cctc.202000512
Perz, F., Bormann, S., Alcalde, M., Hollmann, F., Holtmann, D. and Liese. A. (2020). Enzymatic oxidation of butane to 2-butanol in a bubble column. ChemCatChem. 12: 3666-3669. https://doi.org/10.1002/cctc.202000431
Yoon, J., Kim, J., Tieves, F., Zhang, W., Alcalde, M., Hollmann, F. and Park, C.B. (2020). Piezobiocatalysis: Ultrasound-Driven Enzymatic Oxyfunctionalization of C-H Bonds. ACS Catalysis 10: 5236-5242. https://doi.org/10.1021/acscatal.0c00188
Van Shie, M.M.C.H., Kaczmarek, A.T., Tieves, F., Gomez de Santos, P., Paul, C.E., Arends, I.W.C.E., Alcalde, M., Schwarz, G. and Hollmann, F. (2020). Selective oxyfunctionalisation reactions driven by sulfite oxidase-catalysed in situ generation of H2O2. ChemCatChem 12: 1-5. https://doi.org/10.1002/cctc.201902297
Willot, S.J.P., Hoang, M.D., Paul, C.E., Alcalde, M., Arends, I.W.C.E., Bommarius, A.S., Bommarius, B. and Hollmann, F. (2020). FOx News: Towards metanol-driven biocatalytic oxyfunctionalisation reactions. ChemCatChem 12: 2713-2716. https://doi.org/10.1002/cctc.202000197
Vicente, A.I., Viña-Gonzalez, J., Mateljak, I., Monza, E., Lucas, F., Guallar, V. and Alcalde M. (2020). Enhancing thermostability by modifying flexible surface loops in an evolved high-redox potential laccase. AiChE journal 66e:16747. Special issue Founders tribute to the Nobel Prize in Chemistry 2018. https://doi.org/10.1002/aic.16747
Yayci, A., Gomez Baraibar, A., Krewing, M., Fernandez Fueyo, E., Hollmann, F., Alcalde, A., Kourist, R., Bandow, J.E. (2020). Plasma-driven in situ production of hydrogen peroxide for biocatalysis. ChemSusChem 13: 2072-2079. https://doi.org/10.1002/cssc.201903438
Li Y, Ma Y, Li P, Zhang X, Ribitsch D, Alcalde M, Wang Y, Hollmann F. (2020). Enantioselective Sulfoxidation of Thioanisole by Cascading a Choline Oxidase and a Peroxygenase in the presence of Natural Deep Eutectic Solvents. ChemPlusChem 85: 254-257. https://doi.org/10.1002/cplu.201900751
Viña-Gonzalez, J., Martinez, A.T., Guallar, V. and Alcalde, M. (2020). Sequential oxidation of 5-hydroxymethylfurfural to furan-2,5-dicarboxylic acid by an evolved aryl-alcohol oxidase. BBA Proteins and Proteomics 1868: 140293. https://doi.org/10.1016/j.bbapap.2019.140293
2019
Freakley, S.J.,Kochius, S., van Marwijk, J., Fenner, C., Lewis, R.J., Baldenius, K., Marais, S.S., Opperman, D.J., Harrison, S.T.L., Alcalde, M., Smit, M.S. and Hutchings, G.J. (2019). A chemo-enzymatic oxidation cascade to activate C–H bonds with in situ generated H2O2. Nature Communications 10: 1-8. https://doi.org/10.1038/s41467-019-12120-w
Mateljak, I., Monza, E., Lucas, M.F., Guallar, V., Aleksejeva, O., Ludwig, R., Leech, D., Shleev, S. and Alcalde, M. (2019). Increasing redox potential, redox mediator activity and stability in a fungal laccase by computer-guided mutagenesis and directed evolution. ACS Catalysis 9: 4561-4572. https://doi.org/10.1021/acscatal.9b00531
Ma, Y., Li, Y., Ali, S., Li, P., Zhang, W., Rauch, M.C.R., Willot, S.J.P., Ribtisch, D., Choi, Y.H., Alcalde, M., Hollmann, F. and Wang, Y. (2019). Natural deep eutetic solvents as performance additivies for peroxygenase catalysis. ChemCatChem. 11: 1-7.http://dx.doi.org/10.1002/cctc.201901842
Aleksejeva, O., Mateljak, I., Ludwig, R., Alcalde, M. and Shleev, S. (2019). Electrochemistry of a high redox potential laccase obtained by computer-guided mutagenesis combined with directed evolution. Electrochemistry Communications 106: 106511.https://doi.org/10.1016/j.elecom.2019.106511
Mateljak, I., Rice, A., Yang, K., Tron, T. and Alcalde, M. (2019). The generation of thermostable fungal laccase chimeras by SCHEMA-RASPP structure-guided recombination in vivo. ACS Synthetic Biology 8: 833-843. JOURNAL COVER. https://pubs.acs.org/doi/10.1021/acssynbio.8b00509
Viña-Gonzalez, J., Jimenez-Lalana, D., Sancho, F., Serrano, A., Martinez, A.T., Guallar, V. and Alcalde, M. (2019). Structure-guided evolution of aryl alcohol oxidase from Pleurotus eryingii for the selective oxidation of secondary benzyl alcohols. Advanced Synthesis and Catalysis 361: 2514-2525. https://doi.org/10.1002/adsc.201900134
van Schie, M.M.C.H., Zhang, W., Tieves, F., Choi, S.D., Park, C.B., Burek, B.O., Bloh,J., Arends, I.W.C.E., Paul, C.E., Alcalde, M. and Hollmann, F. (2019). Cascading g-C3N4and peroxygenases for selective oxyfunctionalization reactions. ACS Catalysis 9: 7409-7417.https://doi.org/10.1021/acscatal.9b01341
Rauch, M.C.R., Tieves, F., Paul, C.E., Arends, I.W.C.E., Alcalde, M. and Hollmann, F. (2019). Peroxygenase-catalysed epoxidation of styrene derivatives in neat reaction media. ChemCatChem 11: 4519-4523. https://doi.org/10.1002/cctc.201901142
Burek, B.O., de Boer, S.R. Tieves, F., Zhang, W., van Schie, M., Bormann, S., Alcalde, M., Holtmann, D., Hollmann, F., Bahnemann, D.W. and Bloh, J.Z. (2019). Photoenzymatic hydroxylation of ethylbenzene catalyzed by unspecific peroxygenase: origin of enzyme inactivation and the impact of light intensity and temperature. ChemCatChem 11: 3093-3100. https://doi.org/10.1002/cctc.201900610
Barruetabeña, N., Alonso-Lerma, B., Galera-Prat, A., Joudeh, N., Barandiaran, L., Platero, A.I., Aldazabal, L., Garcia-Parra, P., Fertin, M., Manteca, A., Arbulu, M., Alcalde, M., De Sancho, D., Carrion-Vazquez, M. and Perez-Jimenez, R. (2019). Resurrection of a highly efficient Precambrian endoglucanase. Communications Chemistry 2: 76.https://doi.org/10.1038/s42004-019-0176-6
Garcia-Arellano, H., Gonzalez-Alonso, J.L., Ubilla, C., Comelles, F., Alcalde, M., Bernabé, M., Parra, J.L., Ballesteros, A.O. and Plou, F.J. (2019). Production and surfactant properties of tert-butyl a-D-glucopyranosides catalyzed by cyclodextrin glucanotransferase. Catalysts 9: 575. https://doi.org/10.3390/catal9070575
Tieves, F., Willot, S.J.P., van Schie, M.M.C.H., Rauch, M.C.R., Younes, S.H.H., Zhang, W., Dong, J., Gomez de Santos, P., Robbins, J.M., Bommarius, B., Alcalde, M., Bommarius, A.S. and Hollmann, F. (2019). Formate oxidase (FOx) from Aspergillus oryzae: one catalyst to promote H2O2-dependent biocatalytic oxidation reactions. Angewandte Chemie-International Edition 58: 1-6. https://doi.org/10.1002/anie.201902380
Molina-Espeja, P., Santos-Moriano, P., Garcia-Ruiz, E., Ballesteros, A., Plou, F.J. and Alcalde, M. (2019). Structure-guided immobilization of an evolved unspecific peroxygenase. International Journal of Molecular Science 20: 1627.https://www.mdpi.com/1422-0067/20/7/1627
Gomez de Santos, P., Cervantes, F.V., Tieves, F., Plou, F.J., Hollmann, F. and Alcalde, M. (2019). Benchmarking of laboratory evolved unspecific peroxygenases for the synthesis of human drug metabolites. Tetrahedron 75: 1827-1831. https://doi.org/10.1016/j.tet.2019.02.013
Serrano, A., Sancho, F., Viña-Gonzalez, J., Carro, J., Alcalde, M., Guallar, V. and Martinez, A.T. (2019). Switching the substrate preference of fungal aryl-alcohol oxidase: towards stereoselective oxidation of secondary benzyl alcohols. Catalysis Science and Technology 9: 833-841.https://doi.org/10.1039/c8cy02447b
Tieves, F., Tonin, F., Fernandez-Fueyo, E., Robbins, J.M., Bommarius, B., Bommarius, A.S., Alcalde, M. and Hollmann, F. (2019). Energising the E-factor: The E+ -factor. Tetrahedron 75: 1311-1314. https://doi.org/10.1016/j.tet.2019.01.065
Willot, S.J.-P., Fernandez-Fueyo, E., Tienes, F., Pesic, M., Alcalde, M., Arends, I.W.C.E., Park, C.B. and Hollmann, F. (2019). Expanding the spectrum of light-driven peroxygenase reactions. ACS Catalysis 9: 890-894. https://doi.org/10.1021/acscatal.8b03752
Pesic, M., Willot, S., Fernandez-Fueyo, E., Tieves, F., Alcalde, M., and Hollmann, F. (2019). Multienzymatic in situ hydrogen peroxide generation cascade for peroxygenase-catalysed oxyfunctionalisation reactions. Zeitschrift für Naturforschung C 74(3–4)c: 101–104. https://doi.org/10.1515/znc-2018-0137
2018
Ramirez-Escudero, M., Molina-Espeja, P., Gomez de Santos, P., Hofrichter, M., Sanz-Aparicio, J., and Alcalde, M. (2018). Structural insights into de substrate promiscuity of a laboratory-evolved peroxygenase. ACS Chemical Biology. 13: 3259-3268. https://doi.org/10.1021/acschembio.8b00500
Martin-Diaz, J., Paret, C., Garcia-Ruiz, E., Molina-Espeja, P. and Alcalde, M. (2018). Shuffling the neutral drift of unspecific peroxygenase in Saccharomyces cerevisiae. Applied and Environmental Microbiology. 84: e00808-18. JOURNAL COVER AND SPOTLIGHT.https://doi.org/10.1128/AEM.00808-18
Gomez de Santos, P., Cañellas, M., Tieves, F., Younes, S.H.H., Molina-Espeja, P., Hofrichter, M., Hollmann, F., Guallar, V. and Alcalde, M. (2018). Selective synthesis of the human drug metabolite 5´-hydroxypropranolol by and evolved self-sufficient peroxygenase. ACS Catalysis 8: 4789-4799. https://doi.org/10.1021/acscatal.8b01004
Gomez-Fernandez, B., Garcia-Ruiz, E., Martin, J., Gomez de Santos, P., Santos-Moriano, P., Plou, F.J., Ballesteros, A., Garcia, M., Rodriguez, M., Risso, V.A., Sanchez-Ruiz, J.M., Whitney, S.M. and Alcalde, M. (2018). Directed –in vitro- evolution of Precrambrian and extant Rubiscos. Scientific Reports 8: 5532. https://doi.org/10.1038/s41598-018-23869-3
Viña-Gonzalez, J., Elbl, K., Ponte, X., Valero, F.and Alcalde, M. (2018). Functional expression of aryl-alcohol oxidase in Saccharomyces cerevisiae and Pichia pastoris by directed evolution. Biotechnology & Bioengineering. 115: 1666-1674. https://doi.org/10.1002/bit.26585
Carro, J., Fernandez-Fueyo E., Fernandez-Alonso, C., Cañada, J., Ullrich, R., Hofrichter, M., Alcalde, M., Ferreira, P. and Martinez, A. T. (2018). Self-sustained enzymatic cascade for the production of 2,5-furandicarboxylic acid from 5-methoxymethylfurfural. Biotechnology for Biofuels 11: 86. https://doi.org/10.1186/s13068-018-1091-2
Zhang, W., Fernandez-Fueyo, E., Ni, Y., van Schie, M., Gacs, J., Renirie, R., Wever, R., Mutti, F.G., Rother, D., Alcalde, M. and Hollmann, F. (2018). Selective aerobic oxidation reactions via combination of photocatalytic water oxidation and enzymatic oxyfunctionalizations. Nature Catalysis 1: 55-62. https://doi.org/10.1038/s41929-017-0001-5
Gonzalez-Perez, D. and Alcalde M. (2018) .The making of versatile peroxidase by directed evolution. Biocatalysis & Biotransformation 36: 1-11 (for the special issue of biocatalysis in Spain). http://dx.doi.org/10.1080/10242422.2017.1363190
2017
Zhang, W., Burek, B.O., Fernandez-Fueyo, E., Alcalde, M., Bloh, J.Z. and Hollmann, F. (2017). Selective activation of C-H bonds by cascading photochemistry with biocatalysis. Angewandte Chemie-International Edition 56: 15451-15455. SELECTED HOT PAPER. http://dx.doi.org/10.1002/anie.201708668
Mateljak, I., Tron, T. and Alcalde, M. (2017). Evolved alpha-factor prepro-leaders for directed laccase evolution in Saccharomyces cerevisiae. Microbial Biotechnology 10: 1830-1836. http://dx.doi.org/10.1111/1751-7915.12838
Martinez, A.T., Ruiz-Dueñas, F.J., Camarero, S., Serrano, A., Linde, D., Lund, H., Vind, J., Tovborg, M., Herold-Majumdar, O.M., Hofrichter, M., Liers, C., Ullrich, R., Scheibner, K., Sannia, G., Piscitelli, A., Pezzella, C., Sener, M.E., Kılıç, S., van Berkel, W.J.H., Guallar, V., Lucas, M.F., Zuhse, R., Ludwig, R., Hollmann, F., Fernández-Fueyo, E., Record, E., Faulds, C.B., Tortajada, M., Winckelmann, I., Rasmussen, J.A., Gelo-Pujic, M., Gutierrez, A., del Rio, J.C., Rencoret, J. and Alcalde, M. (2017). Oxidoreductases on their way to industrial biotransformations. Biotechnology Advances 35: 815-831. http://dx.doi.org/10.1016/j.biotechadv.2017.06.003
Mate, D.M. and Alcalde, M. (2017). Laccase: a multi-purpose biocatalyst at the forefront of biotechnology. Microbial Biotechnology 10: 1457-1467. https://dx.doi.org/10.1111/1751-7915.12422
Molina-Espeja, P., Gomez de Santos, P., and Alcalde, M. (2017). Directed evolution of unspecific peroxygenase. In: Directed Enzyme Evolution: Advances and Applications. Alcalde, M. (ed). Springer, Switzerland. 127-143.
Mate, D.M., Palomino, M.A., Molina-Espeja, P., Martin-Diaz, J. and Alcalde, M. (2017). Modification of the peroxygenative:peroxidative activity ratio in the unspecific peroxygenase from Agrocybe aegerita by structure-guided evolution.Protein Engineering Design and Selection 30: 189-196. JOURNAL COVER. http://doi.org/10.1093/protein/gzw073
Alcalde, M. (2017). When directed evolution met ancestral enzyme resurrection. Microbial Biotechnology 10:22-24. https://dx.doi.org/10.1111/1751-7915.12452
2016
Molina-Espeja, P., Cañellas, M., Plou, F.J., Hofrichter, M., Lucas, F., Guallar, V. and Alcalde, M. (2016). Synthesis of 1-naphthol by a natural peroxygenase engineered by directed evolution. ChemBioChem 17: 341-349. http://doi.org/10.1002/cbic.201500493
Ni, Y., Fernandez-Fueyo, E., Gomez-Baraibar, A., Ullrich, R., Hofrichter, M., Yanase, H., Alcalde, M., van Berkel, W.J.H. and Hollman, F. (2016). Peroxygenase-catalysed oxyfunctionalization reactions promoted by the complete oxidation of methanol. Angewandte Chemie-International Edition 55: 798-801. http://doi.org/10.1002/anie.201507881
Molina-Espeja, P., Viña-Gonzalez, J., Gomez, B.J., Martin-Diaz, J., Garcia-Ruiz, E. and Alcalde, M. (2016). Beyond the outer limits of nature by directed evolution. Biotechnology Advances 34: 754-767. http://doi.org/10.1016/j.biotechadv.2016.03.008
Gonzalez-Perez, D., Mateljak, I., Garcia-Ruiz, E., Ruiz-Dueñas, F.J., Martínez, A.T. and Alcalde, A. (2016). Alkaline versatile peroxidase by directed evolution. Catalysis Science and Technology 6: 6625-6636. http://doi.org/10.1039/C6CY01044J
Vicente, A.I., Viña-Gonzalez, J., Santos-Moriano, P., Marquez-Alvarez, C., Ballesteros, O. and Alcalde, M. (2016). Evolved alkaline fungal laccase secreted by Saccharomyces cerevisiae as useful tool for the synthesis of C-N heteropolymeric dye. Journal of Molecular Catalysis B: Enzymatic 134: 323-330. http://dx.doi.org/10.1016/j.molcatb.2016.10.004
Saez-Jimenez, V., Acebes, S., Garcia-Ruiz, E., Romero, A., Guallar, V., Alcalde, M., Medrano, F.J., Martínez, A.T. and Ruiz-Dueñas, F.J. (2016). Unveiling the basis of alkaline stability of an evolved versatile peroxidase.Biochemical Journal 473: 1917-1928. http://doi.org/10.1042/BCJ20160248
Fernandez-Fueyo, E., Ni, Y., Gomez Baraibar, A., Alcalde, M., van Langen, L.M. and Hollman, F. (2016). Towards preparative peroxygenase-catalyzed oxyfunctionalization reactions in organic media. Journal of Molecular Catalysis B: Enzymatic 134: 347-352. http://dx.doi.org/10.1016/j.molcatb.2016.09.013
Viña-Gonzalez, J., Gonzalez-Perez, D. and Alcalde, M. (2016). Directed evolution method in Saccharomyces cerevisiae: Mutant library creation and screening. Journal of Visualized Experiments 110: e53761. http://doi.org/10.3791/53761 and see the experiments at http://www.jove.com/video/53761.
Mate, D.M., Gonzalez-Perez, D., Mateljak I., Gomez de Santos, P. Vicente, A.I. and Alcalde, M. (2016). The pocket manual of directed evolution: Tips and tricks. In: Biotechnology of Microbial Enzymes: Production, Biocatalysis and Industrial Applications.Brahmachari, Demain and Adrio Eds. Elsevier. 185-214.
Mate, D.M. and Alcalde, M. (2016). Directed evolution of fungal laccases: An update. In: Advances in Genome Science, Vol 4.Rahman Ed. Bentham Science Publisher. 91-112. http://doi.org/10.2174/9781681081731116040007
2015
Viña-Gonzalez, J., Gonzalez-Perez, D., Ferreira, P., Martinez, A.T. and Alcalde, M. (2015). Focused directed evolution of aryl-alcohol oxidase in yeast by using chimeric signal peptides. Applied and Environmental Microbiology 81: 6451-6462. http://doi.org/10.1128/AEM.01966-15
Molina-Espeja, P., Ma, S., Mate, D.M., Ludwig, R. and Alcalde, M. (2015). Tandem-yeast expression system for engineering and producing unspecific peroxygenase. Enzyme Microbial Technology 73-74:29-33. http://dx.doi.org/10.1016/j.enzmictec.2015.03.004
Alcalde, M. (2015). Engineering the ligninolytic enzyme consortium. Trends in Biotechnology 33: 155-162. http://dx.doi.org/10.1016/j.tibtech.2014.12.007
Mate, D.M. and Alcalde, M. (2015). Laccase engineering: From rational design to directed evolution. Biotechnology Advances 33: 25-40. http://dx.doi.org/10.1016/j.biotechadv.2014.12.007
2014
Gonzalez-Perez, D., Garcia-Ruiz, E., Ruiz-Dueñas, F.J., Martinez, A.T. and Alcalde, M. (2014). Structural determinants of oxidative stabilization in an evolved versatile peroxidase. ACS-Catalysis 4: 3891-3901. http://dx.doi.org/10.1021/cs501218v
Falk, M., Alcalde, M., Bartlett, P., De Lacey, A., Gorton, L., Gutierrez-Sanchez, C., Haddad, R., Leech, D., Ludwig, R., Magner, E., Mate, D. Conghaile, P.O., Ortiz, R., Pita, M., Pöller, S., Ruzgas, T., Salaj-Kosla, U., Schuhmann, W., Sebelius, F., Shao, M., Stoica, L., Tilly, J., Toscano, M.D., Vivekananthan, J., Wright E. and Shleev S. (2014). Self-powered wireless carbohydrate/oxygen sensitive biodevice based on radio signal transmission. PlOS ONE 9: e109104. http://dx.doi.org/10.1371/journal.pone.0109104
Gonzalez-Perez, D. and Alcalde, M. (2014). Assembly of evolved ligninolytic genes in Saccharomyces cerevisiae. Bioengineered 5: 254-263. http://dx.doi.org/10.4161/bioe.29167
Garcia-Ruiz, E., Mate, D.M., Gonzalez-Perez, D., Molina-Espeja, P., Camarero, S., Martínez, A.T., Ballesteros, A.O. and Alcalde, M. (2014). Directed evolution of ligninolytic oxidoreductases: from functional expression to stabilization and beyond. In: Cascade Biocatalysis, Riva and Fessner Eds. Wiley-VCH. 1-18. http://dx.doi.org/10.1002/9783527682492.ch1
Martinez, A.T., Ruiz-Dueñas, F.J., Gutierrez, A., del Rio, J.C., Alcalde, M., Liers, C., Ullrich, R., Hofrichter, M., Scheibner, K., Kalum, L., Vind, J. and Lund, H. (2014). Search, engineering and applications of new oxidative biocatalysts. Biofuels, Bioproducts and Biorefining 8: 819-835. http://dx.doi.org/10.1002/bbb.1498
Kunamneni, A., Plou, F.J., Alcalde, M. and Ballesteros, A. (2014). Trichoderma enzymes for food industries. In: Biotechnology and Biology of Trichoderma. Gupta, V.K., Schmoll, M. Herrera-Estrella, A., Upadhyay, R.S., Druzhinina, I., Tuohy, M.G. Elsevier. 339-344. ISBN: 978-0-444-59576-8.
https://www.elsevier.com/books/biotechnology-and-biology-of-trichoderma/gupta/978-0-444-59576-8
Molina-Espeja, P., Garcia-Ruiz, E., Gonzalez-Perez, D., Ullrich, R., Hofrichter, M. and Alcalde, M. (2014). Directed evolution of unspecific peroxygenase from Agrocybe aegerita. Applied & Environmental Microbiology 80: 3496-3507.
http://dx.doi.org/10.1128/AEM.00490-14
Pita, M., Mate, D.M., Gonzalez-Perez, D., Shleev, S., Fernández, V.M., Alcalde, M. and De Lacey, A.L. (2014). Bioelectrochemical oxidation of water. Journal of the American Chemical Society 136: 5892-5895.
http://dx.doi.org/ 10.1021/ja502044j
Gonzalez-Perez, D., Molina-Espeja, P., Garcia-Ruiz, E. and Alcalde, M. (2014). Mutagenic organized recombination process by homologous in vivo grouping (MORPHING) for directed enzyme evolution. PlOS ONE 9, 3: e90919.
http://dx.doi.org/10.1371/journal.pone.0090919
2013
Pardo, I., Chanaga, X., Vicente, A., Alcalde, M. and Camarero, S. (2013). New colorimetric assays for the directed evolution of fungal laccases to improve the conversion of plant biomass. BMC Biotechnology 13: 90.
http://dx.doi.org/10.1186/1472-6750-13-90
Torres-Salas, P. Mate, D.M., Ghazi, I., Plou, F.J., Ballesteros A.O. and Alcalde M. (2013). Widening the pH activity profile of a fungal laccase by directed evolution. ChemBioChem 14: 934-937.
SELECTED PAPER AS SPOTLIGHT in Angew. Chem. Int. Ed. 2013, 52: 6568-6570.
http://dx.doi.org/10.1002/cbic.201300102
Mate, D., Gonzalez-Perez, D., Kittl, R., Ludwig, R. and Alcalde M. (2013). Functional expression of a blood tolerant laccase in Pichia pastoris.BMC Biotechnology 13: 38.
http://dx.doi.org/10.1186/1472-6750-13-38
Sygmund, C., Santner, P., Krondorfer, I. Peterbauer, C.K., Alcalde, M., Nyanhongo, G.S., Guebitz, G.M., and Ludwig, R. (2013). Semi-rational engineering of cellobiose dehydrogenase for improved hydrogen peroxide production. Microbial Cell Factories 12: 38.
http://dx.doi.org/10.1186/1475-2859-12-38
De Abreu, M., Alvaro-Benito, M., Sanz, J., Plou, F.J., Fernandez-Lobato, M. and Alcalde, M. (2013). Synthesis of 6-kestose using a highly efficient β-fructofuranosidase engineered by directed evolution. Advanced Synthesis and Catalysis 355: 1703-1708.
SELECTED PAPER for the special issue to be distributed at the Biotrans2013.
http://dx.doi.org/10.1002/adsc.201200769
Mate, D., Gonzalez-Perez, D., Falk, M., Kittl, R., Pita, M., De Lacey, A.L., Ludwig, R., Shleev, S. and Alcalde M. (2013). Blood tolerant laccase by directed evolution. Chemistry & Biology 20: 223-231.
http://dx.doi.org/10.1016/j.chembiol.2013.01.001
Mate, D., Garcia-Ruiz, E., Camarero, S., Shubin, V., Falk, M., Shleev, S. and Alcalde, M. (2013). Switching from blue to yellow: Altering the spectral properties of a high redox potential laccase by directed evolution. Biocatalysis & Biotransformation 31: 8-21.
http://dx.doi.org/10.3109/10242422.2012.749463
Fernandez-Arrojo, L., Rodriguez-Colinas, B., Gutierrez-Alonso, P., Fernandez-Lobato, M., Alcalde, M., Ballesteros, A.O. and Plou, F.J. (2013). Dried alginate-entrapped enzymes (DALGEEs) and their application to the production of fructooligosaccharides. Process Biochemistry 48: 677-682.
http://dx.doi.org/10.1016/j.procbio.2013.02.015
2012
Pardo, I., Vicente, A.I., Mate, D.M., Alcalde, M. and Camarero, S. (2012). Development of chimeric laccases by directed evolution. Biotechnology and Bioengineering 109: 2978-2986. http://dx.doi.org/10.1002/bit.24588
Gonzalez-Perez, D., Garcia-Ruiz, E. and Alcalde M. (2012). Saccharomyces cerevisiae in directed evolution: an efficient tool to improve enzymes. Bioengineered 3: 1-6. http://dx.doi.org/10.4161/bbug.19544. ISSUE HIGHLIGHT in Bioengineered.
Camarero, S., Pardo, I., Cañas, A.I., Molina, P., Record, E., Martínez, A.T., Martínez, M.J. and Alcalde, M. (2012). Engineering platforms for directed evolution of laccase from Pycnoporus cinnabarinus. Applied & Environmental Microbiology 78: 1370-1384. http://dx.doi.org/10.1128/AEM.07530-11
Alcalde, M. (2012). Fundamentos de evolución molecular dirigida de enzimas. In: Biocatálisis aplicada a la obtención de fármacos y productos de alto valor añadido. Monografía XXXV de la Real Academia Nacional de Farmacia. Sanchez-Montero J.M, Ortiz de Apodaca, O.; Doadrio, A. eds. 289-316. ISBN: 978-84-938172-7-5. http://www.analesranf.com/index.php/mono/article/view/1320/1378
Garcia-Ruiz, E., Gonzalez-Perez, D., Ruiz-Dueñas, F.J., Martinez, A.T. and Alcalde, M. (2012). Directed evolution of a temperature, peroxide and alkaline pH tolerant versatile peroxidase. Biochemical Journal 441: 487-498. http://dx.doi.org/10.1042/BJ2011119
2011
Torres-Salas, P., Monte-Martínez, B., Cutiño-Avila, B., Rodriguez-Colinas, M., Alcalde, M., Ballesteros, A. and Plou, F.J. (2011). Immobilized biocatalysts: novel approaches and Tools for binding enzymes to supports. Advances Materials 23: 5275-5282. http://dx.doi.org/10.1002/adma.201101821
De Abreu, M.A., Alvaro-Benito, M., Plou, F.J., Fernández-Lobato, M. and Alcalde, M. (2011). Screening beta-Fructofuranosidases Mutant Libraries to Enhance the Transglycosylation Rates of β-(2-6) Fructooligosaccharides. Combinatorial Chemistry & High-throughput Screening 14: 730-738. http://dx.doi.org/10.2174/138620711796504370
Mate, D., García-Ruiz, E., Camarero, S. and Alcalde, M. (2011). Directed Evolution of Fungal Laccases. Current Genomics 12: 113-122. http://dx.doi.org/10.2174/138920211795564322
2010
Mate, D., García-Burgos, C., García, E., Ballesteros, A., Camarero, S. and Alcalde, M. (2010). Laboratory evolution of high redox potential laccases. Chemistry & Biology 17: 1030-1041. PAPER SELECTED FOR PREVIEW in Chemistry & Biology: Sayut, DJ and Sun, L (2010) Creating designer laccases. Chemistry & Biology, 27: 918-920. http://dx.doi.org/10.1016/j.chembiol.2010.07.010
García, E., Maté, D., Ballesteros, A., Martínez, A.T. and Alcalde, M. (2010). Evolving thermostability in mutant libraries of ligninolytic oxidoreductases expressed in yeast. Microbial Cell Factories 9: 17. http://dx.doi.org/10.1186/1475-2859-9-17
Alcalde, M. (2010). Mutagenesis protocols in Saccharomyces cerevisiae by in vivo overlap extension. In: In vitro Mutagenesis Protocols, 3rd ed. Methods in Molecular Biology 634. (Bramman J. ed) Totowa, New Jersey: Springer-Humana Press. 3-15. ISBN: 978-1-60761-651-1. http://dx.doi.org/10.1007/978-1-60761-652-8_1
2008
Zumarraga, M., Vaz Dominguez, C., Camarero, S., Shleev, S., Polaina, J., Martínez-Arias, A., Ferrer, M., de Lacey, A., Fernández, V., Ballesteros, A., Plou, F.J. and Alcalde, M. (2008). Combinatorial Saturation Mutagenesis of the Myceliophthora thermophila Laccase T2 mutant: the Connection between the C-Terminal Plug and the Conserved 509VSG511Tripeptide. Combinatorial Chemistry & High-Throughput Screening 11: 807-816. http://dx.doi.org/10.2174/138620708786734235
Zumarraga, M., Camarero, S., Martinez-Arias, A., Ballesteros, A., Plou, F.J. and Alcalde, M. (2008). Altering the laccase functionality by in vivo assembly of mutant libraries with different mutational spectra. Proteins 71: 250-260. http://dx.doi.org/10.1002/prot.21699
Kunamneni, A., Ghazi, I., Camarero, S., Ballesteros, A., Plou, F.J. and Alcalde, M. (2008). Decolorization of synthetic dyes by laccase immobilized on epoxy-activated carriers. Process Biochemistry 43: 169-178. http://dx.doi.org/10.1016/j.procbio.2007.11.009
Kunamneni, A., Camarero, S., García, C., Plou, F.J., Ballesteros, A. and Alcalde, M. (2008). Engineering and applications of fungal laccases for organic sinthesis. Microbial Cell Factories 7, 32: 1-17. http://dx.doi.org/10.1186/1475-2859-7-32
Ferrer, M., Beloqui, A., Zumárraga, M., Alcalde, M. and Golyshin, P.N. (2008). Microbes and enzymes: recent trends and new directions to expand protein space. In: Protein Engineering Handbook. Eds: Stefan Lutz, Uwe T. Bornscheuer; Wiley-VCH Verlag GMBH & Co; 1: 233-264. http://dx.doi.org/10.1002/9783527634026.ch9
Kunamneni, A., Plou, F.J., Ballesteros, A. and Alcalde, M. (2008). Laccase and their applications: a patent review. Recent Patents on Biotechnology 2: 10-24. http://dx.doi.org/10.2174/187220808783330965
Plou, F.J., Alcalde, M., Ghazi, I., Fernández-Arrojo, L. and Ballesteros, A. (2008). Efficient fructooligosaccharide synthesis with a fructosyltransferase from Aspergillus aculeatus. In: Modern Biocatalysis. Eds: Fessner WD, and Anthonsen T. Wiley-VCH, Weinheim, Germany. 153-171. ISBN: 978-3-527-32071-4. http://dx.doi.org/10.1002/9783527623839.ch11
2007
Zumarraga, M., Bulter, T., Shleev, S., Polaina, J., Martínez-Arias, A.,Plou, F.J., Ballesteros, A. and Alcalde, M. (2007). In vitro evolution of a fungal laccase in high concentrations of organic cosolvents. Chemistry & Biology 14: 1052-1064. http://dx.doi.org/10.1016/j.chembiol.2007.08.010
Cañas, A.I., Alcalde, M., Plou, F.J., Martinez, M.J., Martinez, A.T. and Camarero, S. (2007). Transformation of polycyclic aromatic hydrocarbons by laccase is strongly enhanced by phenolic compounds present in soil. Environmental Science and Technology 47: 2964-2971. http://dx.doi.org/10.1021/es062328j
Kunamneni, A., Ballesteros, A., Plou, F.J. and Alcalde, M. (2007). Fungal laccase: a versatile enzyme for biotechnological applications. In: Communicating Current Research and Educational Topics and Trends in Applied Microbiology Ed: Mendez-Vilas. Microbiology Book Series, Formatex Publishers, Spain: 233-245. ISBN: 978-84-611-9422-3. http://www.formatex.org/microbio/pdf/Pages233-245.pdf
Zumarraga, M., Plou, F.J., Garcia, H., Ballesteros, A. and Alcalde, M. (2007). Bioremediation of Polycyclic Aromatic Hydrocarbons by a fungal Laccases engineered by directed evolution. Biocatalysis and Biotransformation 25: 219-228. http://dx.doi.org/10.1080/10242420701444272
Alcalde, M., Ferrer, M. and Plou, F.J. (2007). Environmental biocatalysis: from remediation with enzymes to novel green processes. Biocatalysis and Biotransformation 25: 113. http://dx.doi.org/10.1080/10242420701444397
Alcalde, M. (2007). Laccase: biological functions, molecular structure and industrial applications. In: Industrial Enzymes: structure, function and applications. Eds: J. Polaina J. and A.P. MacCabe. Springer, New York: 459-474. ISBN: 978-1-4020-5376-4. http://dx.doi.org/10.1007/1-4020-5377-0_26
Fernández-Arrojo, L., Marín, D., Gómez de Segura, A., Linde, D., Alcalde, M., Gutiérrez-Alonso, P., Ghazi, I., Plou, F.J., Fernández-Lobato, M. and Ballesteros, A. (2007). Transformation of maltose into prebiotic isomaltooligosaccharides by a novel a-glucosidase from Xantophyllomyces dendrorhous. Process Biochemistry 42: 1530-1536. http://dx.doi.org/10.1016/j.procbio.2007.08.007
2006
Alcalde, M., Zumarraga, M., Polaina, J., Ballesteros, A. and Plou, F.J. (2006). Combinatorial saturation mutagenesis by in vivo overlap extension for the engineering of fungal laccases. Combinatorial Chemistry and High-throughput Screening 9: 719-727. http://dx.doi.org/10.2174/138620706779026079
Alcalde, M., Ferrer, M., Plou, F.J. and Ballesteros A. (2006). Environmental biocatalysis: from remediation with enzymes to novel green processes. Trends in Biotechnology 24: 281-287. http://dx.doi.org/10.1016/j.tibtech.2006.04.002
Beloqui, A., Pita, M., Polaina, J., Martinez, A., Golyshina, O., Zumarraga, M., Yakimov, M., Garcia, H., Alcalde, M., Fernández, V., Elborough, K., Andreu, J., Ballesteros, A., Plou, F.J., Timmis, K., Ferrer, M. and Golyshin, P. (2006). Novel polyphenol oxidase mined from a metagenome expression library of bovine rumen. Journal of Biological Chemistry 281: 22933-22942. http://dx.doi.org/10.1074/jbc.M600577200
Mencía, M., Martínez, A., Alcalde, M. and de Lorenzo, V. (2006). Identification of a gamma-hexachlorocyclohexane dehydrochlorinase (LinA) variant with improved expression and solubility properties. Biocatalysis and Biotransformation 24: 223-230. http://dx.doi.org/10.1080/10242420600667809
Ghazi, I., Fernández-Arrojo, L., Gómez de Segura, A., Alcalde, M., Plou, F.J. and Ballesteros, A. (2006). Beet Sugar Syrup and Molasses as Low-Cost Feedstock for the Enzymatic Production of Fructo-oligosaccharides. Journal of Agricultural and Food Chemistry 54: 2964-2968. http://dx.doi.org/10.1021/jf053023b
Gómez de Segura, A., Alcalde, M., Bernabé, M., Ballesteros, A. and Plou, F.J. (2006). Synthesis of methyl alpha-D-glucooligosaccharides by entrapped dextransucrase from Leuconostoc mesenteroides B-1299. Journal of Biotechnology 124: 439-445. http://dx.doi.org/10.1016/j.jbiotec.2005.12.031
Ballesteros, A., Plou, F.J., Alcalde, M., Ferrer, M., García-Arellano, H., Reyes-Duarte, D. and Ghazi, I. (2006). Enzymatic synthesis of sugar esters and oligosaccharides from renewable resources. In: Biocatalysis in the Pharmaceutical and Biotechnology Industries, Ed: R. Patel. CRC Press, New York: 465-490. ISBN: 978-0-8493-3732-1. http://www.crcpress.com/product/isbn/9780849337321
2005
Reyes-Duarte, D., Polaina, J., López-Cortés, N., Alcalde, M., Plou, F.J., Elborough, K., Ballesteros, A., Timmis, K.N., Golyshin, P.N. and Ferrer, M. (2005). Conversion of a carboxylesterase into a triacylglycerol lipase by random mutation. Angewandte Chemie-International Edition 44: 7553-7557. http://dx.doi.org/10.1002/anie.200502461
Alcalde, M., Bulter, T., Zumárraga, M., García, H., Mencía, M., Plou, F.J. and Ballesteros, A. (2005). Screening mutant libraries of fungal laccases in the presence of organic solvents. Journal of Biomolecular Screening 10: 624-631. http://dx.doi.org/10.1177/1087057105277058
Ghazi, I., Gómez de Segura, A., Fernández-Arrojo, L., Alcalde, M., Yates, M., Rojas-Cervantes, M.L., Plou, F.J. and Ballesteros, A. (2005). Immobilisation of fructosyltransferase from Aspergillus aculeatus on epoxy activated Sepabeads-EC for the synthesis of fructo-oligosaccharides. Journal of Molecular Catalysis B: enzymatic 35: 19-27. http://dx.doi.org/10.1016/j.molcatb.2005.04.013
2004
Gómez de Segura, A., Alcalde, M., López-Cortés, N., Plou, F.J. and Ballesteros, A. (2004). Modulating the synthesis of dextran with the acceptor reaction using native and encapsulated dextransucrases. Food Technology & Biotechnology 42: 337-342.
UDC 547.458.1:577.152.6
García-Arellano, H., Alcalde, M. and Ballesteros, A. (2004). Use and improvement of microbial redox enzymes for environmental purposes. Microbial Cell Factories 3:10-13. http://dx.doi.org/10.1186/1475-2859-3-10
Alcalde, M., Farinas, E.T. and Arnold, F.H. (2004). Colorimetric high-throughput assay for alkene epoxidation catalyzed by cytochrome P450 BM-3 variant 139-3. Journal of Biomolecular Screening 9:141-146. http://dx.doi.org/10.1177/1087057103261913
Farinas, E.T., Alcalde, M. and Arnold, F.H. (2004). Alkene epoxidation catalysed by cytochrome P450 BM-3 mutant 139-3. Tetrahedron 60: 525-528. http://dx.doi.org/10.1016/j.tet.2003.10.099
Gómez de Segura, A., Alcalde, M., Yates, M., Rojas-Cervantes, M.L., López-Cortés, N., Ballesteros, A. and Plou, F.J. (2004). Immobilization of dextransucrase from Leuconostoc mesenteroides NRRL B-512F on Eupergit C supports. Biotechnology Progress 20: 1414-1420. http://dx.doi.org/10.1021/bp0400083
Devulapalle, KS, Gómez de Segura, A, Ferrer, M, Alcalde, M, Mooser, G and Plou, F.J. (2004). Effect of carbohydrate fatty acid esters on Streptococcus sobrinus and glucosyltransferase activity. Carbohydrate Research 339: 1029-1034. http://dx.doi.org/10.1016/j.carres.2004.01.007
Martín, M.T., Cruces, M.A.,Alcalde, M., Plou, F.J., Bernabé M. and Ballesteros, A. (2004). Synthesis of maltooligosylfructofuranosides catalysed by immobilized cyclodextrin glucosyltransferase using starch as donor. Tetrahedron 60: 529-534. http://dx.doi.org/10.1016/j.tet.2003.10.113
2003
Bulter, T., Alcalde, M., Sieber, V., Meinhold, P., Schlachtbauer, C., and Arnold, F.H. (2003). Functional expression of a fungal laccase in Saccharomyces cerevisiae by directed evolution. Applied Environmental Microbiology 69: 987-995. http://dx.doi.org/10.1128/AEM.69.2.987-995.2003
Alcalde, M. (2003). Evolución Molecular Dirigida. Investigación y Ciencia. (Spanish version of Scientific American) 326:69-77.
Bulter, T. and Alcalde, M. (2003) Preparing libraries in Saccharomyces cerevisiae. In: Directed Evolution Library Creation. Methods and Protocols. Methods in Molecular Biology Vol 231. Eds: F.H. Arnold and G. Gergiou. Humana Press, Totowa, New Jersey: 17-22. ISBN: 1-58829-285-1. http://dx.doi.org/10.1385/1-59259-395-X:17
Bulter, T., Sieber, V. and Alcalde, M. (2003) Screening mutant libraries in Saccharomyces cerevisiae. In: Directed Enzyme Evolution. Screening and Selection Methods. Methods in Molecular Biology Vol 230. Eds: F.H. Arnold and G. Gergiou. Humana Press, Totowa, New Jersey: 99-108. ISBN: 1-58829-286-X. http://dx.doi.org/10.1385/1-59259-396-8:99
Alcalde, M. and Bulter, T. (2003) Colorimetric assays for screening laccases. In: Directed Enzyme Evolution. Screening and Selection Methods. Methods in Molecular Biology Vol 230. Eds: F.H. Arnold and G. Gergiou. Humana Press, Totowa, New Jersey: 193-202. ISBN: 1-58829-286-X. http://dx.doi.org/10.1385/1-59259-396-8:193
Alcalde, M., Plou, F.J., Pérez, M., García-Arellano, H., Valdés, I., Méndez, E. and Ballesteros, A. (2003). Chemical modification of carboxylic residues in a cyclodextrin glucanotransferase and its implication in the hydrolysis/transglycosylation ratio of the alpha-amylase family. Journal of Molecular Catalysis: B Enzymatic 26: 57-67. http://dx.doi.org/10.1016/S1381-1177(03)00166-8
Gómez de Segura, A., Alcalde, M., Plou, F.J., Remaud-Simeon, M., Monsan, P. and Ballesteros, A. (2003). Encapsulation in LentiKats of dextransucrase from Leuconostoc mesenteroides NRRL B-1299, and its effect on product selectivity. Biocatalysis and Biotransformation 21: 325-331. http://dx.doi.org/10.1080/10242420310001630191
M.T. Martín, Plou, F.J., Alcalde, M. and Ballesteros, A. (2003).Immobilization on Eupergit C of cyclodextrin glucosyltransferase (CGTase) and properties of the immobilized biocatalyst. Journal of Molecular Catalysis B: Enzymatic 21: 299-308. http://dx.doi.org/10.1016/S1381-1177(02)00264-3
2002
Alcalde, M., Bulter, T. and Arnold, F.H. (2002). Colorimetric assays for biodegradation of polycyclic aromatic hydrocarbons by fungal laccases. Journal of Biomolecular Screening 7: 537-543. http://dx.doi.org/10.1177/1087057102238629
Sun, L., Bulter, T., Alcalde, M., Petrounia, I.P. and Arnold, F.H. (2002). Modification of Galactose Oxidase to Introduce Glucose 6-Oxidase Activity. ChemBioChem 8: 781-783. http://dx.doi.org/10.1002/1439-7633(20020802)3:8<781
Plou, F.J., Martín, M.T.,Gómez de Segura, A., Alcalde, M. and Ballesteros, A. (2002). Glycosyltransferases acting on starch or sucrose for the synthesis of oligosaccharides. Canadian Journal of Chemistry 80: 743-752. http://dx.doi.org/10.1139/v02-104
Martín, M.T., Alcalde, M., Plou F.J. and Ballesteros, A. (2002). Covalent immobilization of cyclodextrin glucosyltransferase (CGTase) in activated silica and Sepharose. Indian Journal of Biochemistry and Biophysics 39: 229-234. http://www.ncbi.nlm.nih.gov/pubmed/?term=22908412
2001
Alcalde, M., Plou, F.J., Martín, M.T., Valdés, I., Méndez, E. and Ballesteros, A. (2001). Succinylation of cyclodextrin glycosyltransferase from Thermoanaerobacter sp. 501 enhances its transferase activity using starch as donor. Journal of Biotechnology 86:71-80. http://dx.doi.org/10.1016/S0168-1656(00)00422-3
Martín, M.T., Alcalde, M., Plou, F.J., Dijkhuizen, L. and Ballesteros, A. (2001). Synthesis of malto-oligosaccharides via the acceptor reaction catalysed by cyclodextrin glycosyltransferases. Biocatalysis and Biotransformation 19: 21-35. http://dx.doi.org/10.3109/10242420109103514
2000
Plou, F.J., Alcalde, M. and Ballesteros, A. (2000). Strategies to enhance the stability of industrial enzymes. Bioforum-International Life Sciences and Technology 2/2000: 85-89.
1999
Plou, F.J., Alcalde, M. and Ballesteros, A. (1999). Estabilidad de los Biocatalizadores. Investigación y Ciencia. (Spanish version of Scientific American) 273: 46-55.
Alcalde, M., Plou, F. J., Andersen, C., Martín, M.T., Pedersen, S. and Ballesteros, A. (1999). Chemical modification of lysine side chains of cyclodextrin glycosyltransferase (CGTase) from Thermoanaerobacter causes a shift from CGTase to a-amylase specificity. FEBS Letters 445: 333-337. http://dx.doi.org/10.1016/S0014-5793(99)00134-9
Alcalde, M., Plou, F.J., Gómez de Segura, A., Remaud, M., Willemot, R.M., Monsan, P. and Ballesteros, A. (1999). Immobilization of native and dextran-free dextransucrases from L. mesenteroides NRRL B-512F for the synthesis of glucooliogosaccharides. Biotechnology Techniques 13: 749-755. http://dx.doi.org/10.1023/A:1008966213425
Alcalde, M., Plou, F.J., Gómez de Segura, A. and Ballesteros, A. (1999). Preparación enzimática de oligosacáridos de interés de alimentario. ANS (Alimentación, Nutrición y Salud). Vol 6, nº3: 77-86. ISSN 1136-4815.
Abella, L., Alcalde, M., Estébanez, B., Cortella, A., Alfayate, C. and Ron, E. (1999). Observations on the stomatal complex in ten species of mosses (Pottiaceae, Bryopsida). Journal of Hattori Botanical Laboratory 86: 179-185. ISSN 0073-0912.
1998
Alcalde, M., Plou, F.J., Pastor, E. and Ballesteros, A. (1998). Effect of chemical modification of Cyclodextrin Glycoslytransferase (CGTase) fromThermoanaerobacter sp. on its activity and product selectivity. Annals of the New York Academy of Science 864: 183-187. http://dx.doi.org/10.1111/j.1749-6632.1998.tb10301.x
Alcalde, M.; Plou, F.J.; Martín, M.T.; Remaud, M.; Monsan, P. and Ballesteros, A. (1998). Stability in the presence of organic solvents of dextransucrase from Leuconostoc mesenteroides NRRL B-512F immobilized in calcium-alginate beads. In: Stability and Stabilization of Biocatalysts. Eds.: A. Ballesteros, F.J., Plou,, J.L., Iborra, and P. Halling. Elselvier, Amsterdam: 535-540. ISBN: 0-444-82970-9. http://dx.doi.org/10.1016/S0921-0423(98)80080-4
Plou, F.J., Ferrer,M., Nuero, M.O., Calvo, M.V., Alcalde, M., Reyes, F. and Ballesteros, A. (1998). Analysis of tween 80 as an esterase/lipase substrate for lipolytic activity assay. Biotechnology Techniques 12: 183-186. http://dx.doi.org/10.1023/A:1008809105270
1996
Alcalde, M., Estébanez, B., Abella, L. and Ron, E. (1996). Protonemal development under different culture conditions in Bartramia Hedw.(Musci). Journal of Hattori Botanical Laboratory 79: 107-114. ISSN 0073-0912.