Miguel Alcalde Group


Directed Enzyme Evolution

On the 25th anniversary of the discovery of directed evolution, this book is intended to be a tribute to the 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.

Publications per year

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

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.

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. In press. 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.