Miguel Alcalde Group


Directed Enzyme Evolution

This book was written in 2017 with the occasion of the 25th anniversary of the discovery of directed evolution. The Nobel prize in Chemistry 2018 has been awarded to Prof. Frances Arnold, and more than never,  this book is a 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.

Publications per year

Miguel Alcalde Laboratory
Institute of Catalysis, ICP-CSIC,
Cantoblanco, 28049, Madrid, Spain
Tel: +34 915854806
Fax: +34 915854760

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

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

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 H2O2Nature 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 COVERhttps://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

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 cerevisiaeApplied 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: 4789-4799https://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

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 cerevisiaeMicrobial 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

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

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

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 cerevisiaeBioengineered 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. InCascade 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. InBiotechnology 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.

Molina-Espeja, P., Garcia-Ruiz, E., Gonzalez-Perez, D., Ullrich, R., Hofrichter, M. and Alcalde, M. (2014). Directed evolution of unspecific peroxygenase from Agrocybe aegeritaApplied & Environmental Microbiology 80: 3496-3507. 

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.

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. 

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.

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.

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.

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.

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.

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.

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.

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 cinnabarinusApplied & 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/BJ20111199


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



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



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



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



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



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



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



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



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



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



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.



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.



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



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.


Miguel de Abreu

Manager Business Development, 3P Biopharmaceuticals, Belfast, UK.

Susana Camarero

CSIC Staff Scientist, CIB-CSIC, Madrid, Spain.

Katarina Elbl-Müllhofer

Engineer at Testo Industrial Services, GmbH, Germany.

Layla Fernandez

Group Leader Protein Engineering in Evoxx technologies, GmbH, Germany.

Carlos Garcia-Burgos

Senior Regulatory Affairs Manager at Acino Pharma AG, Switzerland.

Iraj Ghazi

Scientist at Homology Medicines, Inc, Boston, USA.

David Gonzalez-Perez

Postdoc Scientist at Moffit Cancer Center & Research Institute, Florida, USA.

Mehdi Imani

Associate Professor at Urmia University, Iran.

Adinnaraya Kunamneni

PhD researcher at University of Kentucky, Kentucky State, USA.

Diego Jimenez-Lalana

Predoctoral fellow at CNB-CSIC, Madrid, Spain.

Macarena Lopez

Scientist Process Development CMC, Seville, Spain.

Diana Mate

Talent Attraction Researcher at University Autónoma de Madrid, Spain.

Javier Martin-Diaz

Titulado Superior de actividades técnicas y profesionales CSIC, Spain.

Seyed Mohammad Mosthtaghioun

Academic Staff at Yazd University, Iran.

Miguel Arcangel Palomino-Lopez

Biologist at Andujar town hall, Spain.

Alina Roman

Senior Technologist at Lonza, London, UK.

Pamela Torres-Salas

Senior Technologist at Repsol, Madrid, Spain.

Ana Isabel Vicente-Martin

Account manager at Gilson, Madrid, Spain.

Miren Zumarraga

Senior Scientist at Guserbiot, Vitoria, Spain.