Bioprocess engineering and Applied Biocatalysis Research Group (ENG4BIO) (2017 SGR 1462)

Principal investigator: Dr. Francisco Valero Barranco and Dr. Gregorio Álvaro

Contact:
francisco.valero@uab.cat
gregorio.alvaro@uab.cat
Department of Chemical Biological and Environmental l Engineering
Universitat Autònoma de Barcelona
Barcelona, Spain
Phone: +34935813049/935811809
Fax: +34935812013

Group structure
The group comprises researchers from the Department of Chemical, Biological and Environmental Engineering of the UAB and the Institute of Advanced Chemistry of Catalonia (CSIC).
The group has three research laboratories and a fermentation pilot plant unit:

Laboratory of Bioprocess Engineering
Coordinator Dr. Francisco Valero (francisco.valero@uab.cat)

Laboratory of Systems Biology
Coordinator Joan Albiol Sala (joan.albiol@uab.cat)

Laboratory of Applied Biocatalysis
Coordinator Dr. Gregorio Álvaro (gregorio.alvaro@uab.cat)

Fermentation Pilot Plant-Bioprocess Development Unit (+)
Director: Dra. Glòria Gonzàlez (gloria.gonzalez@uab.cat)
Responsible for Quality Insurance and Programming: Dr. Antoni Casablancas (Antoni.casablancas@uab.cat)

Members

Researchers: Dr. Joan Albiol; Dr. Gregorio Álvaro; Dra. Mª Dolors Benaiges; Dra. Glòria Caminal;; Dra. Glòria González; Dr. Pau Ferrer (now at LIST (Luxemburg); ; Dr. José Luis Montesinos; Dr. Francisco Valero.
Post-Doctoral Researchers: Dra. Marina Guillén, Dr. Xavier García, Dr. Peter Sutton
PhD students: Natalia Alcover;  Sergi Monforte;  Jordi Soler; Daniela Valencia, Luis Miguel Vázquez, Miquel Garcia Bofill, Juan José Barrero, Javier Garrigós, Miguel Angel Nieto, Mario Benito, Josu López; Albert Carceller; Albert Fina; Arnau Gasset.

Research fields
The main objective of the group is to develop biotechnological processes to obtain products of interest for the chemical, pharmaceutical, food&beverage and health industries. Thus, the research group develops its activities in the field of the Industrial Biotechnology (or White Biotechnology).
The research interests are focused on i)  microbial production of recombinant proteins and low molecular weight molecules (bulk/fine chemicals) using several renewable raw materials and ii) enzymatic processes of stereoselective synthesis, developing methodologies and strategies to obtain several products:
  • Stereoselective biocatalysts (aldolases, lipases, oxidoreductases and transaminases).
  • Products of interest for the pharmaceutical and health sectors: iminocyclitols, carboxypeptidases inhibitors (PIC), non-steroidal anti-inflammatory, recombinant proteins for therapeutic use (enzymes, antibody fragments, etc.)
  • Products for the chemical and food industry: intermediates of chemical synthesis (chiral amines, alcohols), nutraceutic products, structured lipids, biofuels (biodiesel), biolubricants, etc. 
Laboratory of Bioprocesses Engineering
The selected biological host systems are the prokaryotic system E. coliand the eukaryotic system P. pastoris.  The aim is to develop standardized methodologies to stablish the optimum operational strategies that could be applied to different target proteins.
In order to achieve this objective several disciplines have to be integrated and optimized with the following common objectives for both biological systems:
  • Optimization of the expression system on a genetic level.
  • Standardized methodologies to set up optimal operational strategies for the production of recombinant proteins, mainly in high-cell density fed-batch cultures.
  • Monitoring, modelling and control of the recombinant protein production processes.
  • Integrated and efficient downstream processes for recovering and purification of the product.
  • Scale-up and scale-down.
  • Microbial enzymes production.
  • Production of therapeutic products for animal and/or human use.
Laboratory of Systems Biology
The metabolic engineering of biological systems is mainly focused on microorganisms (yeast and bacteria) and the main goal is the directed improvement of these cell factories for their use in bioprocesses, particularly in recombinant protein production in P. pastoris and in the production of low molecular weight molecules (bulk/fine chemicals) in several yeasts using renewable raw materials, such as glycerol (by-product of the biodiesel synthesis).
 
The research objectives are related to the application of tools, methodologies and principles of Systems Biology and Synthetic Biology in the field of Metabolic Engineering. More specifically, physiological quantitative analysis by means of different analytic platforms or “omics” (transcriptomics, metabolomics, fluxomics), metabolic modelling of cell factories in bioprocess conditions and the knowledge on the design, construction and improvement of producing strains (Synthetic Biology) and/or the optimization of fermentative processes.
This research programme aims to integrate and apply these results in the Bioprocess Engineering context, in close relation to with other research lines of the group.

Laboratory of Applied Biocatalysis
The main objective is the use of selected biocatalysts, mainly aldolases, lipases, aminotransferases and oxidoreductases in enantioselective synthesis. Biocatalysis has been widely developed in the field of known products achieving in this field the best attainments in terms of efficiencies and sustainability in the search of more efficient synthetic processes. Nevertheless, there is not much implementation of Biocatalysis in the production of new innovative molecules for the discovery of new drugs, food & beverage additives, etc.  The group aims to achieve this goal since the selected enzymes allow easy access to chiral complex molecules that are not readily accessible by classic methodologies of organic chemistry. The research group aims to design and operate enzymatic reactors in non-conventional media and to develop enzyme cascade synthesis processes. 
 
The main objectives are:
  • Obtaining iminocyclitols (carbohydrates analogues) with potential therapeutic activity by chemo-enzymatic cascade strategies using non-complex molecules as precursors.
  • Obtaining stable immobilized biocatalysts. Modulation of enzymatic activity.
  • Development of multienzymatic processes.
  • Modelling, optimization and design of enzymatic bioreactors.
  • Production of stereochemically pure products of interest for the pharmaceutical industry. 
  • Production of products of interest for food & beverage industry (nutraceuticals, flavours)
  • Production of products of interest for the fine chemical (chiral amines) and textile (modification of fibres surface) industries.
  • Development of enzymatic processes for second-generation biofuels production.
  • Development of efficient oxidative processes using oxidoreductases.
  • Production of structured lipids (oils and fats modification).
Fermentation Pilot Plant
The Fermentation Pilot Plant offers a wide experience, infrastructure and expertise to develop and optimize bioprocesses based on the use of microorganisms, yeasts, fungus, animal cells and enzymes as biocatalysts. The PPF services are mainly focused on pharmaceutical, chemical, cosmetic and agro food industry, facilitating the implementation of advanced fermentative processes at industrial scale providing competitiveness to these companies.
The PPF activities are developed under strict confidentiality according to the quality service system of the centre. These activities can be classified as follows:
  • Contract Manufacturing Operation (CMO) for industries or public institutions.
  • Development of bioprocesses at laboratory and pilot scale.
  • Advising and training on bioprocesses advising (equipment, bioprocess design, ect)
  • The PPF have the equipment, services and facilities needed to develop the bioprocesses up to pilot scale, such as:
  • Lab-scale bioreactors (2-5L) and pilot-scale bioreactors (50-300L)
  • Tubular centrifuges at pilot scale (15000g) and disk centrifuges at pilot scale (8800g)
  • Equipment for tangential filtration for microfiltration, ultrafiltration and dialysis processes (up to 2,5 m2filtration area)
  • Mechanical cellular disruptor at lab scale and pilot scale (“french-press”).
  • Up to 30” filtration housing
  • Equipment for analysis. Automatic analysers YSI (Yellow Springs) and Y15 (Biosystems), HPLC and HPLC-mass (Waters), ionic chromatography.
  • Equipment for molecular biology techniques (PCR and RT-PCR, microorganisms identification, electrophoresis).
On the PPF website (http://ppf.uab.cat/) more information about the activities and where the PPF has collaborated can be found.

Projects and contracts with companies
• Title: Biotechnological processes based on microbial platforms for the conversion of CO2 from iron&steel industry into commodities and plastics. (BIOCON-CO2)
Funded by: European Union. Horizon 2020. BIOTEC 5 Ref. 761042. 
Participants: Leitat(Coordinator). 17 companies and universities. 
Principal Investigator: Gregorio Álvaro Campos
From 01-01-18 to 31-12-21. Amount: 500.000 €
•Title: Industrial Biotechnology Innovation and Synthetic Biology Accelerator (IBISBA)
Funded by: European Union. Horizon 2020. Ref.730976. 
Participants: Institut national de la recherche agronomique (Coordinator). 15 companies and universities. 
Principal Investigator: Joan Albiol Sala
From 01-12-17 to 30-11-21. Amount: 497.606€
•Title:   Biorefinería del glicerol: Desarrollo de la factoría celular Pichia pastoris para la bioconversión del glicerol crudo en productos de alto valor añadido. GliBioConver
Funded by: Ministerio de Economía y Competitividad CTQ2016-74959-R
From 01/12/2016 to 31/12/2019
Principal Investigator: Dr. Francisco Valero Barranco. Dr. Pau Ferrer Alegre. Amount: 202.000 €
• Title: Intensificación de Procesos Multienzimáticos.
Funded by: Ministerio de Economía y Competitividad (MINECO). Programa Estatal de Investigación, Desarrollo e Innovación Orientada a los Retos de la Sociedad 2014 Ref. CTQ2014-53114-R. Principal Investigator: Gregorio Álvaro Campos and Josep López Santín
From 01-04-15 to 30-03-18. Amount: 183.000 €
• Title: Expanding the industrial use of Robust Oxidative Biocatalysts for the conversion and production of alcohols (ROBOX).
Funded by: European Union. Horizon 2020. BIOTEC 3
Participants: DSM Chemical Technology R&D B.V.(Coordinator). 19 companies and universities. 
Responsible investigator: Gregorio Álvaro Campos
From 01-04-15 to 31-3-19. Amount: 491.283 €
• Title: Pichia pastoriscomo plataforma para la obtención de productos de interés biotecnológico.
Funded by: Cooperación Hispano-Brasileña. PHBP14/00087.
Principal Investigator: Dr. Francisco Valero Barranco.
From 01-03-15 to 01-03-17. Amount: 19.759 €
• Title: Desarrollo integrado de producción enzimática de biodiesel de 2a generación.
Funded by: MINECO CTQ2013-42391-R.
Principal Investigator: Dr. Francisco Valero Barranco y Dr. Pau Ferrer Alegre.
From el 01-10-14 to 30-09-16. Amount: 168.000 €.
• Title: Integrated Process and Cell Refactoring Systems for Enhanced Industrial Biotechnology (IPCRES)
Funded by: ERA-IB (ERA-NET on Industrial Biotechnology)
Participants: University College London, UCL (UK), Jacobs University Bremen (Germany), SilicoLife (Portugal), Technical University of Denmark (Denmark), Ingenza (Scotland), University of Strathclyde (Scotland), BioProdict (The Netherlands), Universitat Autònoma de Barcelona.
Responsible investigator UAB: Dr Pau Ferrer. Coordinator: Dr Darren Nesbeth (UCL)
From 01-04-15 to 31-03-17. Amount: Subcontratación por servicios al grupo UAB: 8.000 €.
• Title: Producción de una lipasa/esterasa recombinante de Candida rugosa.
Funded by: Petrobras (Brasil).
Principal Investigator: Dr. Francisco Valero Barranco and Dra. Denise Freire.
From 01-02-15 to 01-01-16. Amount: 53.000 €
•Title:   Desarrollo integrado de producción enzimática de biodiesel de 2a generación. CTQ2013-42391-R
Funded by: Ministerio de Economía y Competitividad (MINECO).
Principal Investigator:   Dr. Francisco Valero Barranco.Dr. Pau Ferrer Alegre
From:   01-01- 2014 to 31-12-2016. Amount: 168.000 €
•Title    Red de Biotecnología Industrial Integrativa. Red de Excelencia 2015 
Funded by:Ministerio de Economía y Competitividad Bio2015-71824-REDT
Principal Investigator:   Dr. Pau Ferrer Alegre.
From 01/12/2015 to 31/12/ 2017. Amount: 40.000 €
• Title: Fortalecimiento de la línea de Biocatálisis Enzimática. Funded by: Programa de Atracción e Inserción de Capital Humano Avanzado de la Comisión Nacional de Investigación Científica y Tecnológica (CONICYT) del Gobierno de Chile. Participants: Departamento de Ingeniería Química de la UAB and Escuela de Ingeniería Bioquímica de la Pontificia Universidad Católica de Valparaíso. Responsible investigator: Gregorio Álvaro and Andrés Illanes. From 01-07-14 to 01-10-14 and from 01-07-16 to 01-10-16. Amount: 24.632€

Publications (2013-2018)

Bioprocess Engineering
• Viña-González, J., Elbl, K., Ponte, X., Valero, F., Alcalde, M. (2018). ”Functional expression of aryl-alcohol oxidase in Saccharomyces cerevisiae and Pichia pastoris by directed evolution”.Biotechnology and Bioengineering. 115, 1666-1674.
• Ponte, X., Barrigón, J.M., Maurer, M., Mattanovich, D., Valero, F., Montesinos, J.L. (2018).“Towards optimal substrate feeding for heterologous protein production in Pichia pastoris(Komogataella spp) fed-batch processes under PAOX1control: a modelling aided approach”.Journal of Chemical Technology and Biotechnology 93: 3208-3218.
•Barrero, J.J. Casler, J.C.Valero F.,Ferrer P, Glick, B.S. (2018).“An improved secretion signal enhances the secretion of model proteins from Pichia pastoris”.Microbial Cell Factories, 17: 161 (1-13).
• García-Ortega, X., Valero, F., Montesinos-Seguí, J.L. (2017). “Physiological state as transferable operating criterion to improve recombinant protein production in Pichia pastoristhrough oxygen limitation”.Journal of Chemical technology and Biotechnology. 92, 2573-2582.
• AdelantadoN., TarazonaP., GrillitschK., Valero F., Feussner, I.,Daum G.,Ferrer P(2017).“The effect of hypoxia on the lipidome of recombinant Pichia pastoris”. Microbial Cell Factories, 16(86) 1-15 (2017). 
•Macedo J., Lattari F., Machado, A.C., de Castro, A., Volcán, R., Araripe, F., Valero, F., Freire, D. (2017). “Production of recombinant lipase B from Candida antárcticain Pichia pastorisunder control of the promoter PGK using crude glicerol from biodiesel production as carbon source”. Biochemical Engineering Journal. (2017). 118: 123-131.
•Ponte, X., Montesinos-Seguí, J.L., Valero F. (2016). ”Bioprocess efficiency in Rhizopus oryzaelipase production by Pichia pastorisunder the control of PAOX1 is oxygen tension dependent”. Process Biochemistry, 51: 1954-1963.
•D. Calleja, J. Kavanagh, C. de Mas, J. López-Santín (2016). “Simulation and prediction of protein production in fed-batch E. colicultures: an engineering approach”. Biotechnology and Bioengineering 113:772-782. 
• García-Ortega X., Adelantado N., Ferrer P., Montesinos J.L., Valero F. (2016).”A step forward to improve recombinant protein production in Pichia pastoris: From specific growth rate effect on protein secretion to carbon-starving conditions as advanced strategy”.Process Biochemistry. 51:681-691.• García-Ortega X., Reyes C., Montesinos J.L., Valero F. (2016). “Overall key performance indicator to optimizing operation of high-pressure homogenizers for a reliable quantification of intracellular components in Pichia pastoris”.Frontiers in Bioengineering and Biotechnology. 3:107, 1-9.
• Barrigón J.M., Valero F., Montesinos J.L. (2015). A macrokinetic model-based comparative meta-analysis of recombinant protein production by Pichia pastoris under AOX1 promoter. Biotechnology and Bioengineering. 112 (6):1132-1145.
• Pliego J., Mateos J.C., Rodriguez J., Valero F., Baeza M., Femat R., Camacho R., Sandoval G., Herrera-López E.J. (2015). Monitoring lipase/esterase activity by stopped flow in a sequential injection analysis system using p-nitrophenyl butyrate. Sensors. 15(2):2798-2811.
• Calleja D., Fernández-Castañé A., Pasini M., de Mas C., López-Santín J. (2014). Quantitative modeling of inducer transport in fed-batch cultures of E. coli. Biochemical Engineering Journal 91:210-219.
• Hemmerich J., Adelantado N., Barrigón J.M., Ponte X., Hörmann A., Ferrer P., Kensy F., Valero F. (2014). Comprenhensive clone screening and evaluation of fed-batch strategies in a microbioreactor and lab scale stirred tank bioreactor system: application on Pichia pastoris producing Rhizopus oryzae lipase. Microbial Cell Factories. 13:36.
• Barba V., Arnau C., Martínez M.J., Valero F. (2014). Production of a sterol esterase from Ophiostoma piceae in batch and fed-batch bioprocesses using different Pichia pastoris phenotypes as cell factory. Biotechnology Progress 30(5):1012-1020.
• García-Ortega X., Montesinos J.L., Valero F. (2013) Fed-batch operational strategies for recombinant Fab production with Pichia pastoris using the constitutive GAP promoter. Biochemical Engineering Journal 79:172-181.
• Lončar N., Boļić N., López-Santín J., Vujčić Z. (2013). Bacillus amyloquefaciens laccase- From soil bacteria to recombinant enzyme for wastewater decolorization. Bioresource Technology 147:177-183.
• Casablancas A., Cárdenas-Fernández M., Álvaro G., Benaiges M.D., Caminal G., de Mas C., González G., López C., López-Santín J. (2013). New ammonia lyases and amine transaminases: standardization of production process and preparation of immobilized biocatalysts. Electronic Journal of Biotechnology, 16(3):1-13.
• Ruiz J., Fernández-Castañé A., de Mas C., González G., López-Santín J. (2013). From laboratory to pilot plant E. coli fed-batch cultures: optimizing the cellular environment for protein maximization. Journal of Industrial Microbiology and Biotechnology 40:335-343.
• N. Boļić, J.M. Puertas, N. Lončar, C. Sans Duran, J. López-Santín, Z. Vujčić (2013) "The DsbA signal peptide-mediated secretion of a highly efficient raw-starch-digesting, recombinant α-amylase from Bacillus licheniformis ATCC 9945a. Process Biochemistry 48:438-442

Systems Biology
• Tomàs-Gamisans, M., Ferrer, P., Albiol, J. (2017). “Fine-tuning the P. pastorisiMT1026 genome-scale metabolic model for improved prediction of growth on methanol or glycerol as sole carbon sources”. Microbial Biotechnology.11, 224-237. 
• Cámara, E., Landes, N., Albiol, J., Gasser, B., Mattanovich, D., Ferrer, P. (2017). “Increased dosage of AOX1promoter-regulated expression cassettes leads to transcription attenuation of the methanol metabolism in Pichia pastoris”Scientific Reports.7, 44302. 
• Fuentealba, P., Aros, C., Latorre, Y., Martínez, I., Marshall, S., Ferrer, P., Albiol, J., Altamirano, C. (2017). “Genome-scale metabolic reconstruction for the insidious bacterium in aquaculture Piscirickettsia salmonis”Bioresource Technology.223, 105-114.
• E. Cámara, J. Albiol, P. Ferrer (2016). “Droplet Digital PCR-Aided Screening and Characterization of Pichia pastorisMultiple Gene Copy Strains”. Biotechnology and Bioengineering 113:1542-1551
•M.V. Gabarró, S. Gullón, R.L. Vicente, G. Caminal, R.P. Mellado, J. López-Santín (2017). “A Streptomyces lividansSipY deficient strain as a host for protein production: standardization of operational alternatives for model proteins”. Journal of Chemical Technology and Biotechnology 92: 217-223. 
•M. Tomàs-Gamisans, P. Ferrer, J. Albiol (2016).  “Integration and Validation of the Genome-Scale Metabolic Models of Pichia pastoris: A Comprehensive Update of Protein Glycosylation Pathways, Lipid and Energy Metabolism”. Plos One, 11(1): e0148031
•M. Pasini, A. Fernández-Castané, A. Jaramillo, C. de Mas, G. Caminal, P. Ferrer (2015). “Using promoter libraries to reduce metabolic burden due to plasmid-encoded proteins in recombinant Escherichia coli”. New Biotechnology 33 (1).
• Jordà J., Cueto Rojas H., Carnicer M., Wahl A., Ferrer P., Albiol J. (2014). Quantitative metabolomics and instationary 13C-metabolic flux analysis reveals impact of recombinant protein production on trehalose and energy metabolism in Pichia pastoris. Metabolites 4:281-299.
• Jordà J., Santos de Jesus S., Peltier S., Ferrer P., Albiol J. (2014). Metabolic flux analysis of recombinant Pichia pastoris growing on different glycerol/methanol mixtures by iterative fitting of NMR-derived 13C-labelling data from proteinogenic amino acids. New Biotechnology 31:120-132.
• Ferrer P., Albiol J. (2014). 13C-Based metabolic flux analysis of recombinant Pichia pastoris. Methods in Molecular Biology. 1191:291-313.
• Ferrer P., Albiol J. (2014). 13C-Based metabolic flux analysis in yeast: The Pichia pastoris case. Methods in Molecular Biolology. 1152:209-232.
• Vázquez-Lima F., Silva P., Barreiro A., Martínez-Moreno R., Morales P., Quirós M., González R., Albiol J., Ferrer P. (2014). Use of chemostat cultures mimicking different phases of wine fermentations as a tool for quantitative physiological analysis. Microbial Cell Factories. 13:85.
• Saubí N., Gea-Mallorquí E., Ferrer P., Hurtado C., Sánchez-Úbeda S., Eto Y., Gatell J.M., Hanke T., Joseph J. (2014). Engineering new mycobacterial vaccine design for HIV-TB pediatric vaccine vectored by lysine auxotroph of BCG. Molecular Therapy – Methods and Clinical Development. 1:14017.
• Cole J., Ferrer P., Mattanovich D., Archer D. (2013). Recombinant Protein Production 6: A comparative view on host physiology. New Biotechnology. 30:246.
• Jordà J., Suarez C.A., Carnicer M., ten Pierick A., Heijnen J.J., van Gulik W., Ferrer P., Albiol J., Wahl A. (2013). Glucose-methanol co-utilization in Pichia pastoris studied by metabolomics and instationary 13C flux analysis. BMC Systems Biology 7:17.
• Quirós M., Martínez-Moreno R., Albiol J., Morales P., Vázquez-Lima F., Barreiro-Vázquez A., Ferrer P., González R. (2013). Metabolic flux analysis during the exponential growth phase of Saccharomyces cerevisiae in wine fermentations. PloS One. 8(8):e71909.
• Corchero J.L., Gasser B., Resina D., Smith W., Parrilli E., Vázquez F., Abasolo I., Giuliani M., Jäntti J., Ferrer P., Saloheimo M., Mattanovich D., Schwartz S. Jr, Tutino M.L., Villaverde A. (2013). Unconventional microbial systems for the cost-efficient production of high-quality protein therapeutics. Biotechnology Advances. 31:140-153. 
 
Applied Biocatalysis
•Delgove M.A.F., Valencia D, Solé J., Bernaertsa k.v., De Wildemana S.M.A., Guillén M., Álvaro G., (2019). “High performing immobilized Baeyer-Villiger monooxygenase and glucose dehydrogenase for the synthesis of ε-caprolactone derivative”. Applied Catalysis A, General. 572 ,134–141.
•Solé J, Caminal G, Schürmann M, Álvaro G, Guillén M. (2019). “Co-immobilization of a P450 BM3 and glucose dehydrogenase on diferent suports for application as self-sufficient oxidative biocatalyst”. Journal of Chemical Technology and Biotechnology. 94, 244-255.
•Valencia D, Guillén M. Fürst M., López-Santín, Álvaro G. (2018). “An immobilized and highly stabilized self-sufficient monooxygenase as biocatalyst for oxidative biotransformations”. Journal of Chemical Technology and Biotechnology. 93, 985-993.
•Bonet-Ragel, K., López-Pou L., Tutusaus, G., Benaiges, M.D., Valero F. (2018). “Rice husk ash as a potential carrier for the immobilization of lipases applied in the enzymatic production of biodiesel”. Biocatalysis and Biotransformation. 36(2), 151-158.
•Koutinas, M., Yiangou, C., Osorio, N.M., Ioannou, K., Canet, A., Valero, F., Ferreira-Dias S. (2018). “Application of comercial and non-commercial immobilized lipases for biocatalytc production of etyl lactate in organic solvents”. Bioresource Technology, 247, 496-503.
•Costa, C.M., Osório, N.M., Canet, A., Rivera, I., Sandoval, G., Valero, F., Ferreira-Dias S. (2018). “Production of MLM type structured lipids from grapeseed oil catalyzed by non-commercial lipases”.European Journal of Lipid Science and Technology.120, 1-8
•Bonet-Ragel, K., Canet A., Benaiges M.D., Valero F. (2018).“Effect of acyl-acceptor stepwise addition strategy using alperujo oil as a substrate in enzymatic biodiesel synthesis”. Journal of Chemical technology and Biotechnology. 93, 541-547.
•Daniela Valencia, Marina Guillén, Maximilian J. L. J. Fürst, Josep López-Santín, Gregorio Álvaro (2017). “An immobilized and highly stabilized self-sufficient monooxygenase as biocatalyst for oxidative biotransformations”. Journal of Chemical Technology and Biotechnology, 93: 985-993
•Guillén M., Benaiges M.D., Valero F. (2016) “Improved ethyl butyrate synthesis catalyzed by an immobilized recombinant Rhizopus oryzae lipase: A comprehensive statistical study by production, reaction rate and yield analysis”. Journal of Molecular Catalysis B: Enzymatic, 133: S371-S376
• G. Masdeu, S. Kralj, S. Pajk, J. López-Santín, D. Makovec, G.Álvaro (2018).”Hybrid chloroperoxidase-magnetic nanoparticle clústers: effect of functionalization on biocatalyst performance”. Journal of Chemical Technology and Biotechnology 93: 233-245
•Marco Filice, Marta Molina, M. Dolors Benaiges, Olga Abian, Francisco Valero, Jose M. Palomo. (2017). “Solid-surface activated recombinant Rhizopous oryzae  lipase expressed in Pichia pastoris  and chemically modified variants as efficient catalysts in the synthesis of hydroxy monodeprotected glycols”. Catalysis Science & Technology 7, 1766- 1775.
• Albert Canet, Kirian Bonet-Ragel M. Dolors Benaiges, Francisco Valero. (2017). Biodiesel synthesis in a solvent-free system by recombinant Rhizopus oryzae: comparative study between a stirred tank and a packed-bed batch reactor.Biocatalysis and Biotransformation, 35 (1), 35-40. 
•Sandoval G., Casas-Godoy, L., Bonet-Ragel, K., Rodrigues, J., Ferreira-Dias, S., Valero, F. (2017). “Enzyme-catalyzed production of biodiesel as alternative to chemical-catalyzed processes: Advantages and Constraints”. Current Biochemical Engineering4 (2),109-141.
• Canet, A., Benaiges, M.D., Valero, F., Adlercreutz, P. (2017). Exploring substrate specificities of a recombinant Rhizopus oryzae lipase in biodiesel synthesis. New Biotechnology. 39, 59-67. (2017).
 • R.A. Rodríguez-Hinestroza, C. López, J. López-Santín, Ch. Kane, M.D. Benaiges, T. Tzedakis (2017). “HLADH-catalyzed synthesis of β-amino acids, assisted by continuous electrochemical regeneration of NAD+ in a filtre press microreactor”. Chemical Engineering Science 158: 196-207. 
•C. Bahamondes, G. Álvaro, L. Wilson, A. Illanes (2016). “Effect of enzyme load and catalyst particle size on the diffusional restrictions in reactions of synthesis and hydrolysis catalyzed by α-chymotrypsin immobilized in glyoxal agarose”. Process Biochemistry. 
•G. Masdeu, M. Pérez-Trujillo, J. López-Santín, G.Álvaro (2016).” Data on the identification and characterization of by-products from N-Cbz-3-aminopropanal and t-BuOOH/H2O2 chemical reaction in chloroperoxidase-catalyzed oxidations.”. Data in Brief. 8: 659-65.
•G. Masdeu, M. Pérez-Trujillo, J. López-Santín, G.Álvaro (2016).” Data on the identification and characterization of by-products from N-Cbz-3-aminopropanal and t-BuOOH/H2O2 chemical reaction in chloroperoxidase-catalyzed oxidations.”. Data in Brief. 8: 659-65. 
•C. Bahamondes, L. Wilson, C. Bernal,  A. Illanes, G. Álvaro, F. Guzmán (2016). “Synthesis of the Kyotorphin Precursor Benzoyl-L-tyrosine-L-Argininamide with Immobilized  alfa-Chymotrypsin in Sequential Batch with Enzyme Reactivation” Biotechnology Progress. 32:54-59
• Faustino A. R., Osório N. M., Tecelão C., Canet A., Valero F., Ferreira-Dias S. (2016) “Camelina oil as a source of polyunsaturated fatty acids for the production of human milk fat substitutes catalyzed by a heterologous Rhizopus oryzaelipase”. European Journal of Lipid Science and Technology 118:532-544. 
• Canet A., Bonet-Ragel K., Benaiges M.D., Valero F. (2016). “Lipase-catalysed transesterification: Viewpoint of the mechanism and influence of free fatty acids. Biomass and Bioenergy. 85:94-99. • Rodrigues J, Canet A., Rivera I., Osório N.M., Sandoval G., Valero F., Ferreira-Dias S.(2016). “Biodiesel production from crude Jatropa oil catalyzed by non-conventional immobilized heterologous Rhizopus oryzae and Carica papayalipases”. Bioresource Technology. 213: 88-95. 
•Clementz A.L., Del Peso, G., Canet A., Yori J.C., Valero F.(2016). “Utilization of discard bovine bone as a support for immobilization of recombinant Rhizopus oryzae lipase expressed inPichia pastoris”.Biotechnology Progress, 32(5): 1246-1253 (2016).
•Bonet-Ragel K., Canet A., Benaiges M.D., Valero F. (2015). “Synthesis of biodiesel from high alperujo oil catalysed by immobilized lipase”. Fuel. 161:12-17. 
• Hartwig Duarte S., del Peso Hernández G.L., Canet A., Benaiges M.D., Maugeri F., Valero F. (2015). Enzymatic biodiesel synthesis from yeast oil using immobilized recombinant Rhizopus oryzae lipase. Bioresource Technology. 183:175-180.
• Cárdenas-Fernandez M., Khalikova E., Korpela T., López C., Álvaro G. (2015). Co-immobilized aspartase and transaminase for high-yield synthesis of L-phenylalanine. Biochemical Engineering Journal 93: 173-178.
• Quintana P.G., Canet A., Marciello M., Valero F., Palomo J.M., Baldessari A. (2015). Enzyme catalyzed preparation of chenodeoxycholic esters by an immobilized heterologous Rhizopus oryzae lipase. Journal of Molecular Catalysis B: Enzymatic. 118:36-42 (2015).
• Lotti M., Pleiss J., Valero F., Ferrer P. (2015). Effects of methanol on lipases: Molecular, kinetic and process issues in the production of biodiesel. Biotechnology Journal. 10: 22-30.
• Canet A., Benaiges M.D. Valero F (2014). Biodiesel and monoglycerides production using immobilized 1(3)-positional specific recombinant Rhizopus oryzae lipase. Journal of American Oil Chemists’ Society 91:9 1499-1506.
• M. Pešić , N. Boļić , C. López , N. Lončar , G. Álvaro, Z. Vujčić (2014). Chemical modification of chloroperoxidase for enhanced stability and activity. Process Biochemistry 49: 1472-1479.
• Simoes T., Valero F., Tecelao, C., Ferreira-Dias S. (2014). Production of human milk fat substitutes catalyzed by a heterologous Rhizopus oryzae lipase and commercial lipases. Journal of American Oil Chemists’ Society 91:3 411-419.
• Pešić M., López C., Álvaro G., López-Santín J. (2013). From amino alcohol to aminopolyol: one-pot multienzyme oxidation and aldol addition. Applied Microbiology and Biotechnology 97:7173-7183.
• Ferreira-Diaz S., Sandoval G., Plou F., Valero F. (2013). The potential use of lipases in the production of fatty acid derivatives for the food and nutraceutical industries. Electronic Journal of Biotechnology 16:3-5.
• Martínez-Martínez M., Alcaide M., Tchigvintsev A., Reva O., Polaina J., Bargiela R., Guazzaroni N.E., Chicote A., Canet A., Valero F., Eguizaba E.R., Guerrero M.C., Yakunin A.F., Ferrer M. (2013). Biochemical diversity of carboxyl esterases and lipases from Lake Arreo (Spain) – a metagenomic approach. Applied Environmental Microbiology 79(12):3553-3562.
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