Bioprocess Engineering and Applied Biocatalysis Research Group(ENG4BIO) (2017 SGR 1462)
Principal researchers: Francisco Valero Barranco, PhD and Gregorio Álvaro Campos, PhD
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) in Escherichia coli and Pichia pastoris using several renewable raw materials; specially the group is focused on:
- Metabolic engineering for the design and improvement of microbial cell factories.
- Development and intensification of bioprocesses.
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), nutraceuticals, structured lipids, biofuels (biodiesel), biolubricants, etc.
Laboratory of Bioprocesses Engineering
The selected biological host systems are the prokaryotic system E. coli and 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 fibre 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 m2 filtration 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
- Alcover, N., Carceller, A., Álvaro, G., Guillén, M. (2019). “Zymobacter palmae pyruvate decarboxylase production process development: Cloning in Escherichia coli, fed-batch culture and purification”. Engineering in Life Science. 19, 502-512.
- Macedo, J.M. García-Ortega, X. Montesinos-Seguí, J.L. Freire, D.M. Valero, F (2019). Continuous operation, a realistic alternative to fed-batch fermentation for the production of recombinant lipase B from Candida antarctica under the constitutive promoter PGK in Pichia pastoris. Biochemical Engineering Journal. 147:39-47.
- García-Ortega, X., Cámara, E., Ferrer, P., Albiol, J., Montesinos-Seguí, J.L., Valero, F. Rational development of bioprocess engineering strategies for recombinant protein production in Pichia pastoris (Komagataella phaffii) using the methanol-free GAP promoter. Where do we stand? (2019). New Biotechnology 53, 24-34.
- Nieto-Taype, M.A. Garrigós-Martínez, J. Sánchez-Ferrando, M. Valero F., García-Ortega, X. Montesinos-Seguí, J.L. Rational-based selection of optimal operating strategies and gene dosage impact on recombinant protein production in Komagataella phaffi (Pichia pastoris). (2019) Microbial Biotechnology 13, 315-32.
- Garrigós-Martínez, J. Nieto-Taype, M.A. Gasset-Franch, A. Montesinos-Seguí, J.L. García-Ortega, X. Valero, F. Specific growth rate governs AOX1 gene expression, affecting the production kinetics of Pichia pastoris (Komagataella phaffii) PAOX1-driven recombinant producer strains with different target gene dosage. (2019) Microbial Cell Factories. 18, 187.
- Macedo, J.M. Ocampo-Betancurt, M. Oliveira, A.C. Arruda, A. Castelo, V. Volcan,R. Araripe, F. Ferrer, P. Valero, F. Freire, D.M. Increase of Candida antarctica lipase B production under PGK promoter in Pichia pastoris: effect of multicopies. (2019). Brazilian Journal of Microbiology. 50 (2).
- Barrero, J.J. Casler, J.C. Valero F., Ferrer P, Glick, B.S. An improved secretion signal enhances the secretion of model proteins from Pichia pastoris. (2018). Microbial Cell Factories, 17: 161 (1-13).
- •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 PAOX1 control: a modelling aided approach. Journal of Chemical Technology and Biotechnology. 93: 3208-3218.
- Viña-González, J., Elbl, K., Ponte, X., Valero, F., Alcalde, M. Functional expression of aryl-alcohol oxidase in Saccharomyces cerevisiae and Pichia pastoris by directed evolution. (2018).Biotechnology and Bioengineering. 115, 1666-1674.
- García-Ortega, X., Valero, F., Montesinos-Seguí, J.L. (2017). “Physiological state as transferable operating criterion to improve recombinant protein production in Pichia pastoris through oxygen limitation”. Journal of Chemical Technology and Biotechnology. 92, 2573-2582.
- Adelantado N., Tarazona P., Grillitsch K., 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 antárctica in Pichia pastoris under 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 oryzae lipase production by Pichia pastoris under 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. coli cultures: 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.
- Tomàs-Gamisans, M., Andrade, C.C.P., Maresca, F., Monforte, S., Ferrer, P., Albiol, J. Redox engineering by ectopic overexpression of NADH kinase in recombinant Pichia pastoris (Komagataella phaffii): Impact on cell physiology and recombinant production of secreted proteins. (2020) Applied Environmental Microbiology.2,86(6).
- Torres, P., Saa, P.A., Albiol, J., Ferrer, P., Agosín, E.(2019). Contextualized genome-scale model unveils high-order metabolic effects of the specific growth rate and oxygenation level in recombinant Pichia pastoris. Metabolic Engineering Communications. 9, e00103.
- Zahrl, R.J., Gasser, B., Mattanovich, D., Ferrer P. Detection and elimination of cellular bottlenecks in protein-producing yeasts. (2019). Methods in Molecular Biology. 1923, 75-95.
- Cámara, E., Monforte, S., Albiol, J., Ferrer, P. Deregulation of methanol metabolism reverts transcriptional limitations of recombinant Pichia pastoris (Komagataella spp) with multiple expression cassettes under control of the AOX1 promoter (2019). Biotechnology and Bioengineering. 116(7),1710-1720.
- Tomàs-Gamisans, M., Ødum, A.S.R., Workman, M., Ferrer, P., Albiol J. Glycerol metabolism of Pichia pastoris (Komagataella spp.) characterised by 13C-based metabolic flux analysis. (2019). New Biotechnology 50, 52-59.
- Tomàs-Gamisans, M., Ferrer, P., Albiol, J. (2017). “Fine-tuning the P. pastoris iMT1026 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 AOX1 promoter-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.
- Cámara E., Albiol J., Ferrer P. (2016). “Droplet Digital PCR-Aided Screening and Characterization of Pichia pastoris Multiple Gene Copy Strains”. Biotechnology and Bioengineering 113:1542-1551
- Gabarró M.V., Gullón S., Vicente R.L., Caminal G., Mellado R.P., López-SantínJ. (2017). “A Streptomyces lividans SipY deficient strain as a host for protein production: standardization of operational alternatives for model proteins”. Journal of Chemical Technology and Biotechnology 92: 217-223.
- Tomàs-Gamisans M., Ferrer P., Albiol J. (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
- Pasini M., Fernández-Castané A., Jaramillo A., de Mas C., Caminal G., Ferrer P. (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.
- Achouri, N.Tomàs-Gamisans, M. Triki, S. Valero, F. Miled, N. Fendri, A. Smichi, N. (2020). Dissecting the interaction deficiency of a cartilaginous fish digestive lipase with pancreatic colipase: biochemical and structural insights. Biomed Research International (in press).
- Solé, J., Brummund, J., Caminal, G., Álvaro, G., M., Schürman, Guillén, M. ,(2019). “Enzymatic Synthesis of Trimethyl-ϵ-caprolactone: Process Intensification and Demonstration on a 100 L Scale”. Organic Process Research and Development. 23 ,2336-2344.
- Solé, J., Brummund, J., Caminal, G., Schürman, M., Álvaro, G., Guillén, M. ,(2019). “Ketoisophorone Synthesis with an Immobilized Alcohol Dehydrogenase”. ChemCatChem. 11 ,4862-4870.
- Solé, J., Brummund, J., Caminal, G., Schürman, M., Álvaro, G., Guillén, M.,(2019). “Trimethyl-ε-caprolactone synthesis with a novel immobilized glucose dehydrogenase and an immobilized thermostable cyclohexanone monooxygenase”. Applied Catalysis A General. 585, art. Number 117187.
- García-Bofill, M., Sutton, P.W., Guillén, M., Álvaro, G..,(2019). “Enzymatic synthesis of vanillin catalysed by an eugenol oxidase”. Applied Catalysis A General. 582, art. Number 117117.
- 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.
- López-Fernández, J. Barrero, J.J. Benaiges, M.D. Valero, F. (2019) Truncated prosequence of Rhizopus oryzae lipase: key factor for production improvement and biocatalyst stability. Catalyst. 9, 961.
- Valencia, D., Guillén, M., Fürst M., López-Santín J., 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.
- Lotti, M., Pleiss, J., Valero, F., Ferrer, P. Enzymatic Production of Biodiesel: Strategies to Overcome Methanol Inactivation. (2018). Biotechnology Journal (13(5), e1700155.
- Koutinas, M., Yiangou, C., Osorio, N.M., Ioannou, K., Canet, A., Valero, F., Ferreira-Dias S. Application of commercial and non-commercial immobilized lipases for biocatalytic production of ethyl lactate in organic solvents. (2018). Bioresource Technology, 247, 496-503.
- 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.
- Masdeu G., Kralj S., Pajk S., López-Santín J., Makovec D., Álvaro G. (2018).”Hybrid chloroperoxidase-magnetic nanoparticle clústers: effect of functionalization on biocatalyst performance”. Journal of Chemical Technology and Biotechnology 93: 233-245
- Filice M., Molina M., Benaiges M.D., Abian O., Valero F., Palomo JM. (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.
- Canet A., Bonet-Ragel K., Benaiges M.D., Valero F. (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 Engineering. 4 (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.
- Rodríguez-Hinestroza R.A. , López, C., López-Santín, J., Kane, CH., Benaiges, M.D., Tzedakis, T. (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.
- Quintana P.G., Canet A., Marciello M., Valero F., Palomo J.M., Baldessari A. Enzyme catalyzed preparation of chenodeoxycholic esters by an immobilized heterologousRhizopus oryzae lipase. (2015). Journal of Molecular Catalysis B: Enzymatic. 118:36-42.
- Bonet-Ragel K., Canet A., Benaiges M.D., Valero F. Synthesis of biodiesel from high alperujo oil catalysed by immobilized lipase. (2015). Fuel. 161:12-17.
- Faustino A. R., Osório N. M., Tecelão C., Canet A., Valero F., Ferreira-Dias S. Camelina oil as a source of polyunsaturated fatty acids for the production of human milk fat substitutes catalyzed by a heterologous Rhizopus oryzae lipase. (2016). European Journal of Lipid Science and Technology 118:532-544.
- Canet A., Bonet-Ragel K., Benaiges M.D., Valero F. Lipase-catalysed transesterification: Viewpoint of the mechanism and influence of free fatty acids. (2016). Biomass and Bioenergy. 85:94-99.
- Rodrigues J, Canet A., Rivera I., Osório N.M., Sandoval G., Valero F., Ferreira-Dias S. Biodiesel production from crude Jatropa oil catalyzed by non-conventional immobilized heterologous Rhizopus oryzae and Carica papaya lipases. (2016). Bioresource Technology. 213: 88-95.
- Clementz A.L., Del Peso, G., Canet A., Yori J.C., Valero F. Utilization of discard bovine bone as a support for immobilization of recombinant Rhizopus oryzae lipase expressed in Pichia pastoris. (2016). Biotechnology Progress. 32(5), 1246-1253.
- Guillén, M., Benaiges, M.D., Valero, F. Improved ethyl butyrate synthesis catalyzed by an immobilized recombinant Rhizopus oryzae lipase: A comprehensive statiscal study by production, reaction rate and yield analysis. (2016). Journal of Molecular Catalysis B: Enzymatic. 133 S371-S-376.