Papers by Karen Rodríguez
Galactaric acid production by engineering substrate specificity in glucose oxidase from Aspergillus niger
Biochemical Engineering Journal
Sugar oxidation and its products, such as organic acids derived from monosaccharides, have become... more Sugar oxidation and its products, such as organic acids derived from monosaccharides, have become relevant resources following global industrial sustainability concerns. Galactaric acid is a promising renewable chemical building block, potentially replacing fossil-based terephthalic acid derivatives and adding value to pectin-rich biomass. However, novel biocatalysts able to produce galactaric acid from the abundant pectin building block, galacturonic acid, are needed. In this work, glucose oxidase from Aspergillus niger was subjected to protein engineering to modify its substrate specificity away from glucose and towards galacturonic acid for galactaric acid production. The resulting mutagenesis libraries were screened for activity against galacturonic acid, identifying two variants having amino acid substitutions F414K and Y68W. These variants catalyze galacturonic acid conversion in the presence of catalase, with minimal glucose oxidation activity. In silico amino acid substitution analysis in the active site suggests changes in substrate orientation for galacturonic acid, resembling that of glucose in the parental glucose oxidase. Thermal performance and pH stability experiments on the identified variants suggest further structural and functional differences, potentially related to the observed changes in catalytic performance. Finally, in a one-pot reaction with pectinase, the release of galacturonic acid from citrus pectin and its oxidation to galactaric acid was measured by HPLC, showing that simultaneous pectin depolymerization by pectinase and the production of galactaric acid from the released galacturonic acid by the engineered glucose oxidase is possible. This study provides a proof of concept for a potential enzymatic production of hexaric acid building blocks from complex macromolecules such as pectin, using well-established enzymes, such as pectinase preparations and engineered glucose oxidases.
https://www.sciencedirect.com/journal/biocatalysis-and-agricultural-biotechnology, 2022
Current sustainability guidelines require industrial byproducts to be returned to the production ... more Current sustainability guidelines require industrial byproducts to be returned to the production cycle. One of the most important byproducts for agriculture and food production is pectin, which can be hydrolyzed to obtain pectin oligosaccharides (POS), with potential use as prebiotics. One of the challenges in POS production is the modulation of their molecular weight since those between 1 and 3 kDa exhibit higher prebiotic activity. In this work, a pectinase preparation was used for pectin hydrolysis, evaluating the effect of enzyme immobilization on the molecular weight distribution of the obtained POS. The pectinase preparation immobilized on functionalized silica with amino and glyoxyl groups and expressed 88.6 U per g of support. Enzyme immobilization improves thermal resistance at 50 °C and pH 4.0 compared to soluble pectinase. Immobilization also influences pectin hydrolysis and thus the POS generation, increasing yield by 10% when the immobilized pectinase was used. Interestingly, pectinase immobilization resulted in an average of 2.5 kDa for obtained POS.
In contrast, soluble pectinase resulted in a continuous decrease of POS molecular weight over hydrolysis time. This difference could be attributed to the enzyme confinement on the porous silica, limiting the reduction in molecular weight of POS, probably because the immobilized enzyme exhibits higher diffusional restrictions than free enzymes. Our results help to understand and design an efficient and sustainable process to produce tailored prebiotic molecules from agro-industrial byproducts.
Biochemical Engineering Journal, 2022
Sugar oxidation and its products, such as organic acids derived from monosaccharides, have become... more Sugar oxidation and its products, such as organic acids derived from monosaccharides, have become relevant resources following global industrial sustainability concerns. Galactaric acid is a promising renewable chemical building block, potentially replacing fossil-based terephthalic acid derivatives and adding value to pectin-rich biomass. However, novel biocatalysts able to produce galactaric acid from the abundant pectin building block, galacturonic acid, are needed. In this work, glucose oxidase from Aspergillus niger was subjected to protein engineering to modify its substrate specificity away from glucose and towards galacturonic acid for galactaric acid production. The resulting mutagenesis libraries were screened for activity against galacturonic acid, identifying two variants having amino acid substitutions F414K and Y68W. These variants catalyze galacturonic acid conversion in the presence of catalase, with minimal glucose oxidation activity. In silico amino acid substitution analysis in the active site suggests changes in substrate orientation for galacturonic acid, resembling that of glucose in the parental glucose oxidase. Thermal performance and pH stability experiments on the identified variants suggest further structural and functional differences, potentially related to the observed changes in catalytic performance. Finally, in a one-pot reaction with pectinase, the release of galacturonic acid from citrus pectin and its oxidation to galactaric acid was measured by HPLC, showing that simultaneous pectin depolymerization by pectinase and the production of galactaric acid from the released galacturonic acid by the engineered glucose oxidase is possible. This study provides a proof of concept for a potential enzymatic production of hexaric acid building blocks from complex macromolecules such as pectin, using well-established enzymes, such as pectinase preparations and engineered glucose oxidases.
Molecules, 2023
Histamine is a biogenic amine found in fish-derived and fermented food products with physiologica... more Histamine is a biogenic amine found in fish-derived and fermented food products with physiological relevance since its concentration is proportional to food spoilage and health risk for sensitive consumers. There are various analytical methods for histamine quantification from food samples; however, a simple and quick enzymatic detection and quantification method is highly desirable. Histamine dehydrogenase (HDH) is a candidate for enzymatic histamine detection; however, other biogenic amines can change its activity or produce false positive results with an observed substrate inhibition at higher concentrations. In this work, we studied the effect of site saturation mutagenesis in Rhizobium sp. Histamine Dehydrogenase (Rsp HDH) in nine amino acid positions selected through structural alignment analysis, substrate docking, and proximity to the proposed histamine-binding site. The resulting libraries were screened for histamine and agmatine activity. Variants from two libraries (positions 72 and 110) showed improved histamine/agmatine activity ratio, decreased substrate inhibition, and maintained thermal resistance. In addition, activity characterization of the identified Phe72Thr and Asn110Val HDH variants showed a clear substrate inhibition curve for histamine and modified kinetic parameters. The observed maximum velocity (Vmax) increased for variant Phe72Thr at the cost of an increased value for the Michaelis-Menten constant (Km) for histamine. The increased Km value, decreased substrate inhibition, and biogenic amine interference observed for variant Phe72Thr support a tradeoff between substrate affinity and substrate inhibition in the catalytic mechanism of HDHs. Considering this tradeoff for future enzyme engineering of HDH could lead to breakthroughs in performance increases and understanding of this enzyme class.
UNED Research Journal, 2016
Due to the importance of primary production in the trophic network, most aquatic ecosystems rely ... more Due to the importance of primary production in the trophic network, most aquatic ecosystems rely on microalgae. However, there are fewer studies on phytoplankton in the Central American region, specifically on marine diatoms, in part because it is difficult to isolate and cultivate local species in tropical climates. The objective of this study is the isolation of two local diatoms of Chaetoceros and Nitzschia, potential food for aquatic organisms. Water samples was taken from the Nicoya Gulf, located in the Central Pacific coast of Costa Rica. The samples were successively filtered first through a 35µm pore size filter and then through one of 20µm. They were repeatedly isolated using modified Pasteur pipettes, and serial dilutions were done until a monoalgal culture was obtained. Two Bacillariophyceae were isolated: NGN1, genus Nitzschia; and CGN2, genus Chaetoceros. This study is a step toward reducing the importation of phytoplankton on feeding aquatic organisms by providing tech...
Producción de fitoplancton para la investigación y la docencia en larvicultura de camarones, moluscos y peces, 2008-2013 Phytoplankton Production for Research and Teaching in Larviculture Shrimp, Shellfish and Fish, 2008-2013
Durante 2008 al 2013 la produccion de fitoplancton marino fue una actividad miscelanea que apoyo ... more Durante 2008 al 2013 la produccion de fitoplancton marino fue una actividad miscelanea que apoyo la docencia, investigacion y extension. Para ello, anualmente se realizaron mejoras en la produccion masiva, mantenimiento de cepas e intermedios; considerando los factores de tension detectados el ano anterior; cinco productos fundamentales se reportan: 1- adquisicion de 11 especies certificadas, 2- mejoras en protocolos de produccion masiva, 3-aislamiento de dos especies endogenas del Golfo de Nicoya, 4- aclimatacion y escalamiento a 2 Ton de una especie endogena del Golfo de Nicoya clasificada como sp1 y 5- un incremento del 60 % en la produccion de cultivos masivos. Durante este periodo, 10 especies fueron utilizadas como material didactico en 7 cursos de los niveles III y IV de la Carrera de Biologia Marina. Los resultados alcanzados indican que esta actividad ha contribuido al desarrollo de docencia, investigacion y extension que impulsa la Escuela de Ciencias Biologicas. ...
Immobilized Biocatalyst Engineering: High throughput enzyme immobilization for the integration of biocatalyst improvement strategies
International Journal of Biological Macromolecules
The increasing use of sustainable manufacturing technologies in the industry presents a constant ... more The increasing use of sustainable manufacturing technologies in the industry presents a constant challenge for the development of suitable biocatalysts. Traditionally, improved biocatalysts are developed either using protein engineering (PE) or enzyme immobilization (EI). However, these approaches are usually not simultaneously applied. In this work, we designed and validated an enzyme improvement platform, Immobilized Biocatalyst Engineering (IBE), which simultaneously integrates PE and EI, with a unique combination of improvement through amino acid substitutions and attachment to a support material, allowing to select variants that would not be found through single or subsequent PE and EI improvement strategies. Our results show that there is a significant difference on the best performing variants identified through IBE, when compared to those that could be identified as soluble enzymes and then immobilized, especially when evaluating variants with low enzyme as soluble enzymes and high activity when immobilized. IBE allows evaluating thousands of variants in a short time through an integrated screening, and selection can be made with more information, resulting in the detection of highly stable and active heterogeneous biocatalysts. This novel approach can translate into a higher probability of finding suitable biocatalysts for highly demanding processes.
Selective immobilization of Bacillus subtilis lipase A from cell culture supernatant: Improving catalytic performance and thermal resistance
Process Biochemistry
Abstract Bacillus subtilis lipase A (BSLA) has been extensively studied through protein engineeri... more Abstract Bacillus subtilis lipase A (BSLA) has been extensively studied through protein engineering; however, its immobilization and behavior as an insoluble biocatalyst have not been extensively explored. In this work, for the first time, a direct immobilization of recombinant BSLA from microbial culture supernatant was reported, using chemically modified porous with different electrostatic, hydrophobic, hydrophilic, and hydrophilic−hydrophobic enzyme-support interactions. The resulting biocatalysts were evaluated based on their immobilization kinetics, activity expression (pH 7.4), thermal stability (50 °C), solvent resistance and substrate preference. Biocatalysts obtained using glyoxyl silica support resulted in the selective immobilization of BSLA, resulting in an activity recovery of 50 % and an outstanding aqueous stabilization factor of 436, and 9.5 in isopropyl alcohol, compared to the free enzyme. This selective immobilization methodology of BSLA allows to efficiently generate immobilized biocatalysts, thus avoiding laborious purification steps from cell culture supernatant, which is usually a limiting step when large amounts of enzyme variants or candidates are assessed as immobilized biocatalysts. Direct enzyme immobilization from cell supernatant provides an interesting tool which can be used to facilitate the development and assessment of immobilized biocatalysts from engineered enzyme variants and mutant libraries, especially in harsh conditions, such as high temperatures or non-aqueous solvents, or against non-water-soluble substrates. Furthermore, selective immobilization approaches from cell culture supernatant or clarified lysates could help bridging the gap between protein engineering and enzyme immobilization, allowing for the implementation of immobilization steps in high throughput enzyme screening platforms for their potential use in directed evolution campaigns.
Integrating enzyme immobilization and protein engineering: An alternative path for the development of novel and improved industrial biocatalysts
Biotechnology advances, Jan 9, 2018
Enzyme immobilization often achieves reusable biocatalysts with improved operational stability an... more Enzyme immobilization often achieves reusable biocatalysts with improved operational stability and solvent resistance. However, these modifications are generally associated with a decrease in activity or detrimental modifications in catalytic properties. On the other hand, protein engineering aims to generate enzymes with increased performance at specific conditions by means of genetic manipulation, directed evolution and rational design. However, the achieved biocatalysts are generally generated as soluble enzymes, -thus not reusable- and their performance under real operational conditions is uncertain. Combined protein engineering and enzyme immobilization approaches have been employed as parallel or consecutive strategies for improving an enzyme of interest. Recent reports show efforts on simultaneously improving both enzymatic and immobilization components through genetic modification of enzymes and optimizing binding chemistry for site-specific and oriented immobilization. None...
Grasas y Aceites
Microalgae represent an important nutritional source for diverse organisms, therefore, their nutr... more Microalgae represent an important nutritional source for diverse organisms, therefore, their nutritional value, and more specifically, total lipid and fatty acid contents, must be considered. This study evaluated the nutritional contents and potential growth under controlled conditions of Nitzschia sp. and Chaetoceros sp. Tropical microalgae, isolated from the Gulf of Nicoya, Costa Rica. In both strains, the nutritional composition and the fatty acid profile were evaluated in exponential and stationary phases. With regards to fatty acids, v sp. had more Eicosapentaenoic Acid (EPA) in both the exponential (32.80%) and stationary (27.20%) phases. The results in growth rate, production and biochemical composition indicated two tropical microalgae strains suitable for cultivation under controlled conditions. The studies of the phytoplankton in this geographical area is highly relevant because of its importance in the primary production of nutrients and the importance of finding sources ...
SN Applied Sciences
The aim of this study was to link the biotechnological advances made in arid lands, which are kno... more The aim of this study was to link the biotechnological advances made in arid lands, which are known as "brown biotechnology, " to the current environmental challenges facing these regions, their ecosystems and their inhabitants. We present a broad compilation of scientific evidence and technological advances developed over the past 15 years that have helped to address some of the classical constraints of arid environments, such as the development of sustainable agriculture in areas limited by water resources and low levels of organic matter in soils, including the relevant organisms and their interactions with biotic and abiotic stressors. New challenges and new opportunities related to the production of sustainable energy, versatile crops, phytoremediation, biofertilization, desalination, and the incorporation of genes into plants to improve their resistance to water scarcity and to obtain biomolecules from the organisms living in these hostile environments are also presented. To face the constant and recently extreme changes in the climate and the worrisome scarcity of natural resources successfully, issues that need to be resolved in the short and medium terms are described here.
Revista UniveRsidad en diálogo, 2015
From 2008 to 2013 production of marine phytoplankton was a miscellaneous activity that supporter ... more From 2008 to 2013 production of marine phytoplankton was a miscellaneous activity that supporter teaching, investigation and extension. For this, improvements were made annually in the massive production, maintenance of stock and intermediates; considering the factors of tension detected last year, five fundamental products are reported: 1- acquisition of 11 certified species, 2- improvements in massive production protocols, 3- isolation of two endogenous species to the Gulf of Nicoya, 4- acclimation and scaling to 2 Tons of one species endogenous to the Gulf of Nicoya classifies as spl. And 5- a 60% increment in the production of massive cultures. During this period, 10 species were used as didactic material in 7 courses of levels III and IV of the Marine Biology career. The results accomplished indicate that this activity has contributed to the development of teaching, investigation and extension that promotes the School of Biological Sciences. It is important to continue the task of acquiring endemic, acclimated species that are identified and validated as a source of live food, through the biochemical characterization of the nutritional profile in the isolated species.
J Food Sci Technol, 2020
Your article is protected by copyright and all rights are held exclusively by Association of Food... more Your article is protected by copyright and all rights are held exclusively by Association of Food Scientists & Technologists (India). This e-offprint is for personal use only and shall not be self-archived in electronic repositories. If you wish to self-archive your article, please use the accepted manuscript version for posting on your own website. You may further deposit the accepted manuscript version in any repository, provided it is only made publicly available 12 months after official publication or later and provided acknowledgement is given to the original source of publication and a link is inserted to the published article on Springer's website. The link must be accompanied by the following text: "The final publication is available at link.springer.com".
GRASAS Y ACEITES , 2017
Microalgae represent an important nutritional source for diverse organisms, therefore, their nutr... more Microalgae represent an important nutritional source for diverse organisms, therefore, their nutritional value, and more specifically, total lipid and fatty acid contents, must be considered. This study evaluated the nutritional contents and potential growth under controlled conditions of Nitzschia sp. and Chaetoceros sp. Tropical microalgae, isolated from the Gulf of Nicoya, Costa Rica. In both strains, the nutritional composition and the fatty acid profile were evaluated in exponential and stationary phases. With regards to fatty acids, Nitzschia sp. had more Eicosapentaenoic Acid (EPA) in both the exponential (32.80%) and stationary (27.20%) phases. The results in growth rate, production and biochemical composition indicated two tropical microalgae strains suitable for cultivation under controlled conditions. The studies of the phytoplankton in this geographical area is highly relevant because of its importance in the primary production of nutrients and the importance of finding sources of fatty acids such as the EPA.
Research Journal of the Costa Rican Distance Education University, 2016
Due to the importance of primary production in the trophic network, most aquatic ecosystems rely ... more Due to the importance of primary production in the trophic network, most aquatic ecosystems rely on microalgae. However, there are fewer stud- ies on phytoplankton in the Central American region, specifically on marine diatoms, in part because it is difficult to isolate and cultivate local species in tropical climates. The objective of this study is the isola- tion of two local diatoms of Chaetoceros and Nitzschia, potential food for aquatic organisms. Water samples was taken from the Nicoya Gulf, located in the Central Pacific coast of Costa Rica. The samples were suc- cessively filtered first through a 35μm pore size filter and then through one of 20μm. They were repeatedly isolated using modified Pasteur pipettes, and serial dilutions were done until a monoalgal culture was obtained. Two Bacillariophyceae were isolated: NGN1, genus Nitzschia; and CGN2, genus Chaetoceros. This study is a step toward reducing the importation of phytoplankton on feeding aquatic organisms by provid- ing technical and scientific information to develop a microalgal culture from this region.
International Journal of Biological Macromolecules, 2021
The increasing use of sustainable manufacturing technologies in the industry presents a constant ... more The increasing use of sustainable manufacturing technologies in the industry presents a constant challenge for the development of suitable biocatalysts. Traditionally, improved biocatalysts are developed either using protein engineering (PE) or enzyme immobilization (EI). However, these approaches are usually not simultaneously applied. In this work, we designed and validated an enzyme improvement platform, Immobilized Biocatalyst Engineering (IBE), which simultaneously integrates PE and EI, with a unique combination of improvement through amino acid substitutions and attachment to a support material, allowing to select variants that would not be found through single or subsequent PE and EI improvement strategies. Our results show that there is a significant difference on the best performing variants identified through IBE, when compared to those that could be identified as soluble enzymes and then immobilized, especially when evaluating variants with low enzyme as soluble enzymes and high activity when immobilized. IBE allows evaluating thousands of variants in a short time through an integrated screening, and selection can be made with more information, resulting in the detection of highly stable and active heterogeneous biocatalysts. This novel approach can translate into a higher probability of finding suitable biocatalysts for highly demanding processes.
Springer Nature, 2020
The aim of this study was to link the biotechnological advances made in arid lands, which are kno... more The aim of this study was to link the biotechnological advances made in arid lands, which are known as "brown biotechnology, " to the current environmental challenges facing these regions, their ecosystems and their inhabitants. We present a broad compilation of scientific evidence and technological advances developed over the past 15 years that have helped to address some of the classical constraints of arid environments, such as the development of sustainable agriculture in areas limited by water resources and low levels of organic matter in soils, including the relevant organisms and their interactions with biotic and abiotic stressors. New challenges and new opportunities related to the production of sustainable energy, versatile crops, phytoremediation, biofertilization, desalination, and the incorporation of genes into plants to improve their resistance to water scarcity and to obtain biomolecules from the organisms living in these hostile environments are also presented. To face the constant and recently extreme changes in the climate and the worrisome scarcity of natural resources successfully, issues that need to be resolved in the short and medium terms are described here.
Chem. Biodiversity , 2020
The wild edible mushroom ‘digüeñe’ (Cyttaria espinosae) is a mushroom with interesting flavor and... more The wild edible mushroom ‘digüeñe’ (Cyttaria espinosae) is a mushroom with interesting flavor and nutritional properties, but highly perishable with a consumption very limited, generating inevitable economic losses for their producers. Because of that, the aim of this study was the evaluation of two drying methods (hot-air drying and freeze-drying) in its bioactive and physicochemical composition for its preservation. The best performances of antioxidant capacity were 62 μmol TE g−1 (ORAC) and 14.21 μmol TE g−1 (DPPH), being higher compared with other vegetable sources. Its protein (5.33 %), fiber (5.58 %) and K (2.82 mg g−1) content also stands out. β-Carotene retention was 35.02 and 23.25 % after freeze drying and hot-air, respectively. Freeze-drying seems a good strategy for digüeñe, bioactive compounds, color and structure were better maintained. This work represents the first characterization of dehydrated digüeñe, constituting approaches for its valorization and use in the food industry.
Process Biochemistry, 2020
Bacillus subtilis lipase A (BSLA) has been extensively studied through protein engineering; howev... more Bacillus subtilis lipase A (BSLA) has been extensively studied through protein engineering; however, its immobilization and behavior as an insoluble biocatalyst have not been extensively explored. In this work, for the first time, a direct immobilization of recombinant BSLA from microbial culture supernatant was reported, using chemically modified porous with different electrostatic, hydrophobic, hydrophilic, and hydrophilic−hydrophobic enzyme-support interactions. The resulting biocatalysts were evaluated based on their immobilization kinetics, activity expression (pH 7.4), thermal stability (50°C), solvent resistance and substrate preference. Biocatalysts obtained using glyoxyl silica support resulted in the selective immobilization of BSLA, resulting in an activity recovery of 50 % and an outstanding aqueous stabilization factor of 436, and 9.5 in isopropyl alcohol, compared to the free enzyme. This selective immobilization methodology of BSLA allows to efficiently generate immobilized biocatalysts, thus avoiding laborious purification steps from cell culture supernatant, which is usually a limiting step when large amounts of enzyme variants or candidates are assessed as immobilized biocatalysts. Direct enzyme immobilization from cell supernatant provides an interesting tool which can be used to facilitate the development and assessment of immobilized biocatalysts from engineered enzyme variants and mutant libraries, especially in harsh conditions, such as high temperatures or non-aqueous solvents, or against non-water-soluble substrates. Furthermore, selective immobilization approaches from cell culture supernatant or clarified lysates could help bridging the gap between protein engineering and enzyme immobilization, allowing for the implementation of immobilization steps in high throughput enzyme screening platforms for their potential use in directed evolution campaigns.
International Communications in Heat and Mass Transfer, 2019
A numerical study describes the conjugate unsteady natural heat convection of air on top of a non... more A numerical study describes the conjugate unsteady natural heat convection of air on top of a non-Newtonian polymer-water solution characterized by a power-law model within a sealed vertical thick-walled cylindrical enclosure partially filled with a porous media in the lower section. Enzymes immobilized in an inorganic polymer, silica, used in synthesis processes constitute the porous media. Heating is by a constant high temperature at the bottom and at the vertical walls while the upper horizontal wall remains at the initial cold temperature. The Finite Volume Method allows the solution of the continuity, linear momentum and energy equations for a power law shear thinning fluid in a porous media with the Darcy-Brikman-Forchheimer model. The evolution of fluid mechanics of air and of the non-Newtonian fluid is described by the history of the streamlines. Transient heat transfer by conduction in the sealed tube walls and by natural convection of air and in the non-Newtonian fluid flow in the porous media is characterized by the evolution of isotherms. Experimental results are used to verify that the natural convective heat transfer conjugate computational model allows the prediction of temperature evolution in a thermal resistance challenge for free and immobilized enzymes. A difference of 1% is found between the experimental and the numerical results for the temperature history during the immobilized enzyme convective heating process. The applied numerical method has the capability of designing the enzyme temperature challenge test, by predicting the time needed to reach the uniform target temperature for biocatalyst processing, 500 s, in agreement with the experimental findings. The computational model contributes to the development of a technique to describe the kinetics of both immobilized and free enzyme during heating. This is a key issue for several industries, such as food modification, biofuel development, agroindustry and fishery waste valorization, that require enzymes operating at high temperature ranges in a reliable way for a long time.
Uploads
Papers by Karen Rodríguez
In contrast, soluble pectinase resulted in a continuous decrease of POS molecular weight over hydrolysis time. This difference could be attributed to the enzyme confinement on the porous silica, limiting the reduction in molecular weight of POS, probably because the immobilized enzyme exhibits higher diffusional restrictions than free enzymes. Our results help to understand and design an efficient and sustainable process to produce tailored prebiotic molecules from agro-industrial byproducts.