Article
Biochemistry & Molecular Biology
Sara Arana-Pena, Diego Carballares, Roberto Morellon-Sterling, Javier Rocha-Martin, Roberto Fernandez-Lafuente
Summary: This study achieved the coimmobilization of lipases from Rhizomucor miehei and Candida antarctica, successfully immobilizing both enzymes even when one of them cannot be immobilized initially. A special strategy was developed to desorb inactivated enzyme and re-immobilize a fresh sample, allowing for enzyme reuse.
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
(2022)
Article
Biochemistry & Molecular Biology
El-Hocine Siar, Roberto Morellon-Sterling, Diego Carballares, Javier Rocha-Martin, Oveimar Barbosa, Juan M. Bolivar, Roberto Fernandez-Lafuente
Summary: The immobilization of ficin extract on glyoxyl agarose beads stabilized the enzyme, but the stability was highest when immobilized for 3 hours. The biocatalyst prepared for 24 hours was less stable when thermally inactivated in the presence of air. Incubating the thermally inactivated biocatalysts with dithiothreitol allowed for the recovery of almost total activity, with better recovery observed for the 24 hour biocatalyst. This suggests that the faster enzyme inactivation of the 24 hour biocatalyst was due to Cys oxidation.
PROCESS BIOCHEMISTRY
(2023)
Article
Chemistry, Analytical
Dali Wei, Mingwei Li, Fengxiang Ai, Kun Wang, Nuanfei Zhu, Ying Wang, Daqiang Yin, Zhen Zhang
Summary: The cooperation of biocatalysis and chemocatalysis in a catalytic cascade reaction has been widely studied. However, practical applications are hindered by enzyme fragility, poor compatibility between carriers and enzymes, and limited catalytic efficiency. In this study, a biomimetic cascade nanoreactor (GOx@COFs@Os) was developed, integrating glucose oxidase (GOx) and Os nanozyme with covalent organic framework (COF) capsules. The GOx@COFs@Os capsule provided a spacious microenvironment to retain GOx activity and protect it from incompatible environments. The COF capsule also improved substrate affinity and mass transfer efficiency, resulting in significantly improved catalytic efficiency compared to the free cascade system. Moreover, the biomimetic cascade capsule was successfully used for glucose monitoring, glutathione sensing, and bisphenol S detection. This strategy provides a new avenue for improving biocatalytic cascade performance and expanding its applications in various fields.
ANALYTICAL CHEMISTRY
(2023)
Article
Biochemistry & Molecular Biology
Diego Carballares, Javier Rocha-Martin, Roberto Fernandez-Lafuente
Summary: By chemically aminating immobilized TLL, Lecitase can be ionically exchanged and released from the biocatalyst, allowing the utilization of the fully active TLL to build a new combibiocatalyst. However, the release of Lecitase is not possible using high ionic strength solutions alone, requiring the use of octyl-vinylsulfone supports for irreversible immobilization of TLL.
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
(2022)
Article
Biotechnology & Applied Microbiology
Roberto Morellon-Sterling, El-Hocine Siar, Sabrina Ait Braham, Diandra de Andrades, Justo Pedroche, Ma del Carmen Millan, Roberto Fernandez-Lafuente
Summary: Enzymes such as trypsin, chymotrypsin, penicillin G acylase, and ficin extract were stabilized through immobilization on glyoxyl agarose with the addition of different aliphatic compounds containing a primary amine group. The presence of amines was found to decrease the enzyme-support attachment covalently and reduce enzyme stability, with octyl amine having the most significant effect. The steric hindrance caused by aminated compounds interacting with aldehyde groups on the enzyme/support multi-reaction plays a key role in decreasing enzyme stability, even with just 1 mM of aminated compounds.
JOURNAL OF BIOTECHNOLOGY
(2021)
Article
Chemistry, Physical
Thays N. da Rocha, Roberto Morellon-Sterling, Luciana R. B. Goncalves, Juan M. Bolivar, Andres R. Alcantara, Javier Rocha-Martin, Roberto Fernandez-Lafuente
Summary: In this study, MANAE-vinyl sulfone (VS) agarose beads were found to be a better support for immobilizing PGA enzyme compared to agarose-VS beads. The modified beads allowed for rapid and covalent immobilization of PGA, resulting in improved stability compared to low ionic strength VS-agarose.
Article
Biochemistry & Molecular Biology
Sabrina Ait Braham, Roberto Morellon-Sterling, Diandra de Andrades, Rafael C. Rodrigues, El-Hocine Siar, Ali Aksas, Justo Pedroche, Maria del Carmen Millan, Roberto Fernandez-Lafuente
Summary: The presence of Tris during immobilization was found to reduce the stability of enzymes, particularly for immobilized PGA, possibly due to the homogeneous distribution of Lys groups in PGA compared to trypsin and chymotrypsin.
APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
(2021)
Review
Biotechnology & Applied Microbiology
Rafael C. Rodrigues, Angel Berenguer-Murcia, Diego Carballares, Roberto Morellon-Sterling, Roberto Fernandez-Lafuente
Summary: The use of enzyme immobilization can improve enzyme stability by generating favorable enzyme environments, preventing enzyme subunit dissociation, creating more stable enzyme conformations, or enzyme rigidification. Choosing an ideal immobilization protocol and support active groups, as well as utilizing multipoint covalent attachment, can significantly impact the effectiveness of enzyme immobilization.
BIOTECHNOLOGY ADVANCES
(2021)
Article
Biochemistry & Molecular Biology
Diego Carballares, Javier Rocha-Martin, Roberto Fernandez-Lafuente
Summary: Researchers have proposed a new strategy to solve the problem of enzyme inactivation by using three different immobilization protocols, allowing for the reuse of immobilized enzymes and reducing waste.
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
(2022)
Article
Chemistry, Multidisciplinary
Satyadip Paul, Mani Gupta, Kaushik Dey, Ashok Kumar Mahato, Saikat Bag, Arun Torris, E. Bhoje Gowd, Hasnain Sajid, Matthew A. Addicoat, Supratim Datta, Rahul Banerjee
Summary: In this study, a hierarchical TpAzo COF-foam with micro-, meso-, and macroporosity was synthesized using an in situ CO2 gas effervescence technique. The resulting TpAzo foam matrix showed higher immobilization efficiency for enzymes compared to the COF. The enzyme-immobilized TpAzo foam exhibited activity and stability, and was successfully utilized for a one-pot tandem conversion reaction with high recyclability.
Article
Biochemistry & Molecular Biology
Thays N. da Rocha, Roberto Morellon-Sterlling, Javier Rocha-Martin, Juan M. Bolivar, Luciana R. B. Goncalves, Roberto Fernandez-Lafuente
Summary: Penicillin G acylase from Escherichia coli was successfully immobilized on vinyl sulfone agarose beads. The immobilization was optimized with the purpose of obtaining a stable and active biocatalyst. The study found that vinyl sulfone agarose beads have heterofunctionality, offering new possibilities for enzyme immobilization.
Article
Biochemistry & Molecular Biology
Roberto Morellon-Sterling, Juan M. Bolivar, Roberto Fernandez-Lafuente
Summary: The immobilization of ficin on vinyl sulfone agarose caused its almost complete inactivation, possibly due to the modification of catalytic cysteine. By switching off ficin with dipyridyl-disulfide during immobilization and switching it on with 1 M beta-mercaptoethanol, the activity of the immobilized ficin could be significantly increased.
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
(2022)
Article
Biochemistry & Molecular Biology
Shireen A. A. Saleh, Walaa A. Abdel Wahab, Faten A. Mostafa, Marwa I. Wahba
Summary: Amidated pectin-polyethylene imine-glutaraldehyde (AP-PEI-GA) immobilizer with high xylanase activity was prepared and the ideal protocol for its preparation was investigated. Covalent coupling to AP-PEI-GA improved the thermodynamic properties of Aspergillus niger xylanase.
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
(2023)
Article
Chemistry, Multidisciplinary
Ryan S. Czarny, Alexander N. Ho, P. Shing Ho
Summary: Classical hydrogen bonds have long been the main non-covalent interaction used by chemists and chemical engineers to design and control compound structures, but recently non-classical non-covalent interactions such as halogen bonds have also emerged as important tools. Research is focused on understanding the role of halogen bonds in biological systems and utilizing them to control the structures, stabilities, and activities of biomolecules.
Article
Chemistry, Multidisciplinary
Bhabatosh Mandal, Sneha Mondal, Biswajit Hansda, Shailja Mishra, Ankit Ghosh, Tirtha Biswas, Basudev Das, Tanay Kumar Mondal, Pallavi Kumari
Summary: Enzyme immobilization is a crucial technique that improves enzyme stability and activity. In this study, urease was successfully immobilized on three-dimensional silica gel, resulting in a catalyst with high efficiency and reusability. The immobilized enzyme also showed good adaptability and retained its native conformation in different environments.
Article
Biochemistry & Molecular Biology
Diego Carballares, Javier Rocha-Martin, Roberto Fernandez-Lafuente
Summary: Researchers have proposed a new strategy to solve the problem of enzyme inactivation by using three different immobilization protocols, allowing for the reuse of immobilized enzymes and reducing waste.
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
(2022)
Article
Biochemistry & Molecular Biology
Sara Arana-Pena, Diego Carballares, Roberto Morellon-Sterling, Javier Rocha-Martin, Roberto Fernandez-Lafuente
Summary: This study achieved the coimmobilization of lipases from Rhizomucor miehei and Candida antarctica, successfully immobilizing both enzymes even when one of them cannot be immobilized initially. A special strategy was developed to desorb inactivated enzyme and re-immobilize a fresh sample, allowing for enzyme reuse.
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
(2022)
Article
Biochemistry & Molecular Biology
Diego Carballares, Javier Rocha-Martin, Roberto Fernandez-Lafuente
Summary: By chemically aminating immobilized TLL, Lecitase can be ionically exchanged and released from the biocatalyst, allowing the utilization of the fully active TLL to build a new combibiocatalyst. However, the release of Lecitase is not possible using high ionic strength solutions alone, requiring the use of octyl-vinylsulfone supports for irreversible immobilization of TLL.
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
(2022)
Review
Biochemistry & Molecular Biology
Diego Carballares, Roberto Morellon-Sterling, Roberto Fernandez-Lafuente
Summary: This review covers the different possibilities of utilizing enzymes with multiple catalytic activities for cascade reactions. It discusses enzymes with promiscuous activities, fusion enzymes, enzymes combined with metal catalysts, enzymes with non-canonical amino acids and metal catalysts, as well as the design of enzymes with a second biological active center through enzyme modeling and directed mutagenesis. It also highlights the challenges related to balancing catalytic activities and operational stabilities. The design of new multi-activity enzymes shows great potential in establishing new combo-catalysis routes.
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
(2022)
Article
Chemistry, Multidisciplinary
Diego Carballares, Javier Rocha-Martin, Roberto Fernandez-Lafuente
Summary: This study demonstrates a method for the coimmobilization of six different lipases on octyl agarose beads using an enzyme-multilayer strategy and enzyme layer adhesives. The methodology allows for the reuse of stable enzymes after the least stable enzyme inactivation occurs.
ACS SUSTAINABLE CHEMISTRY & ENGINEERING
(2022)
Article
Chemistry, Physical
Thays N. da Rocha, Roberto Morellon-Sterling, Luciana R. B. Goncalves, Juan M. Bolivar, Andres R. Alcantara, Javier Rocha-Martin, Roberto Fernandez-Lafuente
Summary: In this study, MANAE-vinyl sulfone (VS) agarose beads were found to be a better support for immobilizing PGA enzyme compared to agarose-VS beads. The modified beads allowed for rapid and covalent immobilization of PGA, resulting in improved stability compared to low ionic strength VS-agarose.
Article
Chemistry, Physical
Jose R. Guimaraes, Diego Carballares, Javier Rocha-Martin, Andres R. Alcantara, Paulo W. Tardioli, Roberto Fernandez-Lafuente
Summary: The lipase from Thermomyces lanuginosus (TLL) was immobilized on a methacrylate macroporous resin coated with octadecyl groups for enhanced stability. Covalent immobilization of the enzyme was achieved by activating the support with divinyl sulfone. The use of different blocking agents affected the functional features of the biocatalyst.
Article
Biotechnology & Applied Microbiology
Pedro Abellanas-Perez, Diego Carballares, Javier Rocha-Martin, Roberto Fernandez-Lafuente
Summary: This article analyzes the interactions between enzyme crowding and its chemical modification on enzyme activity and stability. The results show that the effects of chemical modifications vary depending on the enzyme support loading. Lowly loaded enzymes increase their activity after modification, while the opposite is true for highly loaded enzymes. TNBS modification increases the stability of highly loaded enzymes but decreases their activity.
BIOTECHNOLOGY PROGRESS
(2023)
Article
Engineering, Chemical
David Gonzalez-Miranda, Diego Carballares, Tomas Pedregal, Roberto Fernandez-Lafuente, Miguel Ladero, Juan M. Bolivar
Summary: This study achieved efficient synthesis of glycerol carbonate using immobilized lipases under solventless conditions. The use of immobilized C. rugosa lipase showed the highest turnover numbers and allowed for multiple reaction cycles. The results of this research highlight the significance of reaction engineering approaches in developing efficient sustainable processes for synthesizing valuable chemicals.
INDUSTRIAL & ENGINEERING CHEMISTRY RESEARCH
(2023)
Article
Biochemistry & Molecular Biology
Pedro Abellanas-Perez, Diego Carballares, Roberto Fernandez-Lafuente, Javier Rocha-Martin
Summary: This study immobilized two lipases, CALB and TLL, on octyl agarose, and observed the stability of the enzymes under different treatment methods and buffers. It was found that the stability of the enzymes varied depending on the treatment method and buffer used. The presence of intermolecular crosslinkings enhanced stability for the highly loaded enzymes, while intramolecular crosslinkings played a role in stabilizing the lowly loaded enzymes.
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
(2023)
Article
Biochemistry & Molecular Biology
El-Hocine Siar, Roberto Morellon-Sterling, Diego Carballares, Javier Rocha-Martin, Oveimar Barbosa, Juan M. Bolivar, Roberto Fernandez-Lafuente
Summary: The immobilization of ficin extract on glyoxyl agarose beads stabilized the enzyme, but the stability was highest when immobilized for 3 hours. The biocatalyst prepared for 24 hours was less stable when thermally inactivated in the presence of air. Incubating the thermally inactivated biocatalysts with dithiothreitol allowed for the recovery of almost total activity, with better recovery observed for the 24 hour biocatalyst. This suggests that the faster enzyme inactivation of the 24 hour biocatalyst was due to Cys oxidation.
PROCESS BIOCHEMISTRY
(2023)
Article
Nanoscience & Nanotechnology
Keyvan Jodeiri, Aleksandra Foerster, Gustavo F. Trindade, Jisun Im, Diego Carballares, Roberto Fernandez-Lafuente, Marcos Pita, Antonio L. De Lacey, Christopher Parmenter, Christopher Tuck
Summary: The drive for miniaturization in enzyme-based bioelectronics has led to the development of 3D microstructured electrodes. Additive manufacturing combined with electroless metal plating allows the production of these electrodes with high surface area. The challenge lies in ensuring strong adhesion between the metal layer and polymer structure, which has been achieved in this study through the introduction of an interfacial adhesion layer.
ACS APPLIED MATERIALS & INTERFACES
(2023)
Article
Chemistry, Multidisciplinary
Leandros Paschalidis, Sara Arana-Pena, Volker Sieber, Jakob Burger
Summary: Advances in enzymatic cascade reactions have led to increased interest in enzyme co-immobilization in porous particles. The optimal conditions for different spatial immobilization distributions (SIDs) are not fully understood, and there is a lack of simulation and optimization methods for these systems. This study provides a theoretical framework for modeling enzymatic cascade reactions with enzymes immobilized in porous particles. Different SIDs were analyzed, and it was found that homogeneous co-immobilization outperforms individual immobilization when diffusion is slow compared to reaction rates. Heterogeneous co-immobilization with enzymes positioned at the entry of the pore is advantageous when reaction rates or substrate diffusion is slow.
REACTION CHEMISTRY & ENGINEERING
(2023)