3D Printing: an Emerging Technology for Biocatalyst Immobilization

Employment of enzymes as biocatalysts offers immense benefits across diverse sectors in the context of green chemistry, biodegradability, and sustainability. When compared to free enzymes in solution, enzyme immobilization proposes an effective means of improving functional efficiency and operational stability. The advance of printable and functional materials utilized in additive manufacturing, coupled with the capability to produce bespoke geometries, has sparked great interest toward the 3-dimensional (3D) printing of immobilized enzymes. Printable biocatalysts represent a new generation of enzyme immobilization in a more customizable and adaptable manner, unleashing their potential functionalities for countless applications in industrial biotechnology. This review provides an overview of enzyme immobilization techniques and 3D printing technologies, followed by illustrations of the latest 3D printed enzyme-immobilized industrial and clinical applications. The unique advantages of harnessing 3D printing as an enzyme immobilization technique will be presented, alongside a discussion on its potential limitations. Finally, the future perspectives of integrating 3D printing with enzyme immobilization will be considered, highlighting the endless possibilities that are achievable in both research and industrySpanish MCI/AEI. Grant Numbers: RTI2018-094482-J-I00, PID2019-105308RB-I00, RYC2018-024846-I. Galician Competitive Research Group (GRC). Grant Numbers: ED431C 2021/29, ED431C 2021/37. Centro Singular de Investigación de Galicia. Grant Number: ED431G 2019/03. European Regional Development FundS

Valorization of bioethanol by-products to produce unspecific peroxygenase with Agrocybe aegerita: Technological and proteomic perspectives

Unspecific peroxygenase (UPO) presents a wide range of biotechnological applications. This study targets the use of by-products from bioethanol synthesis to produce UPO by Agrocybe aegerita. Solid-state and submerged fermentations (SSF and SmF) were evaluated, achieving the highest titers of UPO and laccase in SmF using vinasse as nutrients source. Optimized UPO production of 331 U/L was achieved in 50% (v:v) vinasse with an inoculum grown for 14 days. These conditions were scaled-up to a 4 L reactor, achieving a UPO activity of 265 U/L. Fungal proteome expression was analyzed before and after UPO activity appeared by shotgun mass spectrometry proteomics. Laccase, dye-decolorizing peroxidases (DyP), lectins and proteins involved in reactive oxygen species (ROS) production and control were detected (in addition to UPO). Interestingly, the metabolism of complex sugars and nitrogen sources had a different activity at the beginning and end of the submerged fermentationS.G., A.T. and G.E. thank their grants (BES-2017-081677, FJC2019-041664-I and RYC2018-024846-I, respectively) funded by MCIN/AEI/ 10.13039/501100011033, and by “ERDF A way of making Europe” and “ESF Investing in your future”. Authors would like to thank the use of USC Mass Spectrometry and Proteomics facilities and Bioetanol Galicia S.A. for the supply of the substrates used in the fermentations. The authors belong to the Galician Competitive Research Groups (GRC)_ ED431C-2021/37. The program is co-funded by FEDER (UE)S

Microwave-assisted autohydrolysis of avocado seed for the recovery of antioxidant phenolics and glucose

This study describes the valorization of avocado seed (AS) within a green biorefinery concept using microwave-assisted autohydrolysis. After the treatment at temperatures of 150–230 ◦C for 5 min, the resulting solid and liquor were characterized. The temperature of 220 ◦C led to the simultaneous optimal values of antioxidant phenolics/flavonoids (42.15 mg GAE/g AS, 31.89 RE/g AS, respectively) and glucose + glucooligosaccharides (38.82 g/L) in the liquor. Extraction with ethyl acetate allowed the recovery of the bioactive compounds while maintaining the polysaccharides in the liquor. The extract was rich in vanillin (99.02 mg/g AS) and contained several phenolic acids and flavonoids. The solid phase and the phenolic-free liquor were subjected to enzymatic hydrolysis to produce glucose, reaching values of 9.93 and 105 g glucose/L, respectively. This work demonstrates that microwave-assisted autohydrolysis is a promising technology to obtain fermentable sugars and antioxidant phenolic compounds from avocado seeds following a biorefinery scheme.Universidade de Vigo/CISUGXunta de Galicia | Ref. ED431C 2017/62-GRCXunta de Galicia | Ref. ED431F 2020/03Xunta de Galicia | Ref. ED481B-2022-020Ministerio de Ciencia, Innovación y Universidades | Ref. FPU21/02446Ministerio de Ciencia, Innovación y Universidades | Ref. FJC2021-046978-IMinisterio de Ciencia, Innovación y Universidades | Ref. RYC2018-024846-IMinisterio de Ciencia, Innovación y Universidades | Ref. RYC2018-026177-

Electrochemical oxidation of lignin for the simultaneous production of bioadhesive precursors and value-added chemicals

Electrochemical oxidation of lignin has been widely regarded as a clean and reliable alternative to obtain value-added products from lignin, such as vanillin or guaiacol. This work aims to go one step beyond the production of low molecular weight molecules and explore the possibility of using lignin residues from electrochemical treatments in the context of biorefinery. To this end, a two-way valorization of lignin by electrochemical oxidation is proposed, in order to obtain a liquid phase enriched in low molecular weight organic oligomers and a solid phase of modified lignin to be used as bioadhesive precursor. Hydroxylation of lignin by electrochemical oxidation using boron-doped diamond (BDD) anodes was observed according to the FTIR and MALDI-TOF results, concluding that an applied current density of 10 mA cm−2 leads to promising modifications for the formulation of bioadhesives. Furthermore, NIPU bioadhesives with electrochemically modified lignin were successfully prepared and tested for use in particleboard panels, showing satisfactory mechanical properties, and thus paving the way for more environmentally friendly lignin modification procedures for the wood industryJJC acknowledges financial support from Galician Government though a postdoctoral fellowship (ED481B-2021/015). SG-R and GE predoctoral and postdoctoral fellowships (BES-2017-081677 and RYC-2018-024846-I, respectively) were funded by MCIN/AEI/10.13039/501100011033 and by “ESF Investing in your future”. JJC, SG-R, TAL-C, GE and MTM belong to the Galician Competitive Research Group (GRC) ED431C-2021/37. LERMAB is financed by the French Agence Nationale de la Recherche (ANR) as part of the laboratory of excellence (LABEX) ARBRE. The authors would also like to thank the use of the analytical facilities of IR-Raman Spectroscopy Unit and Mass Spectrometry Unit from RIAIDT-USC.S

Formulation of Laccase Nanobiocatalysts Based on Ionic and Covalent Interactions for the Enhanced Oxidation of Phenolic Compounds

Oxidative biocatalysis by laccase arises as a promising alternative in the development of advanced oxidation processes for the removal of xenobiotics. The aim of this work is to develop various types of nanobiocatalysts based on laccase immobilized on different superparamagnetic and non-magnetic nanoparticles to improve the stability of the biocatalysts. Several techniques of enzyme immobilization were evaluated based on ionic exchange and covalent bonding. The highest yields of laccase immobilization were achieved for the covalent laccase nanoconjugates of silica-coated magnetic nanoparticles (2.66 U mg−1 NPs), formed by the covalent attachment of the enzyme between the aldehyde groups of the glutaraldehyde-functionalized nanoparticle and the amino groups of the enzyme. Moreover, its application in the biotransformation of phenol as a model recalcitrant compound was tested at different pH and successfully achieved at pH 6 for 24 h. A sequential batch operation was carried out, with complete recovery of the nanobiocatalyst and minimal deactivation of the enzyme after four cycles of phenol oxidation. The major drawback associated with the use of the nanoparticles relies on the energy consumption required for their production and the use of chemicals, that account for a major contribution in the normalized index of 5.28 × 10−3. The reduction of cyclohexane (used in the synthesis of silica-coated magnetic nanoparticles) led to a significant lower index (3.62 × 10−3); however, the immobilization was negatively affected, which discouraged this alternativeThis work was financially supported by the Spanish Ministry of Economy and Competitiveness (CTQ2013-44762-R and CTQ2016-79461-R, program co-funded by FEDER). The authors belong to the Galician Competitive Research Group GRC 2013-032, program co-funded by FEDER. Yolanda Moldes-Diz thanks the Spanish Ministry of Economy and Competitiveness for her predoctoral fellowshipS

Integrated biocatalytic platform based on aqueous biphasic systems for the sustainable oligomerization of rutin

Rutin is a known antioxidant compound that displays a broad range of biological activities and health-related benefits but presents a low water solubility that can be overcome by its polymerization. In this work, biocompatible aqueous biphasic systems composed of the ionic liquid cholinium dihydrogen phosphate ([CH][DHph]) and the polymer poly(ethylene glycol) 600 (PEG 600) were investigated as an efficient integrated reaction–separation platform for the laccase-catalyzed oligomerization of rutin. Two different approaches were studied to reuse laccase in several oligorutin production cycles, the main difference between them being the use of monophasic or biphasic regimes during the oligomerization reaction. The use of a biphasic regime in the second approach (heterogeneous reaction medium) allowed the successful reuse of the biocatalyst in three consecutive reaction–separation cycles while achieving noteworthy rutin oligomerization yields (95% in the first cycle, 91% in the second cycle, and 89% in the last cycle). These remarkable results were caused by the combination of the increased solubility of rutin in the PEG-rich phase together with the enhanced catalytic performance of laccase in the [Ch][DHph]-rich phase, alongside with the optimization of the pH of the reaction medium straightly linked to enzyme stability. Finally, a life-cycle assessment was performed to compare this integrated reaction–separation platform to three alternative processes, reinforcing its sustainabilityThis research was supported by the Spanish Government (AEI) through the RTI2018-094482-J-I00 project. This work was developed within the scope of the project CICECO-Aveiro Institute of Materials, UIDB/50011/2020 & UIDP/50011/2020, financed by national funds through the Portuguese Foundation for Science and Technology/MCTES. The programme is cofunded by FEDER (UE). A.P.M.T. thanks the FCT for the research contract CEECIND/2020/01867. G.E. thanks the Spanish MICIU for her Ramón y Cajal contract (RYC2018- 024846-I). A.M.-M. thanks the Programa de axudas á etapa predoutoral da Xunta de Galicia (ED481A-2018/023)S

Tecnologías enzimáticas para la eliminación de compuestos de baja solubilidad en agua

La presencia de compuestos recalcitrantes en efluentes, suelos y sedimentos es un problema medioambiental de especial relevancia. Los compuestos de baja solubilidad tales como hidrocarburos aromáticos policíclicos (HAPs) tienen una alta persistencia ya que, debido a sus propiedades físico-químicas, su degradación por las bacterias autóctonas se ve limitada. Con el objetivo de desarrollar un sistema capaz de llevar a cabo la degradación de estos compuestos, se ha planteado la utilización de los hongos ligninolíticos o bien sus enzimas oxidativas. La enzima manganeso peroxidasa (MnP) promueve la oxidación de Mn+2 a Mn+3, el cual actúa como un poderoso agente oxidante que oxida compuestos orgánicos recalcitrantes de forma inespecífica. La degradación de compuestos de baja solubilidad mediante la enzima MnP requiere la adición de un agente que incremente la solubilidad del compuesto a fin de facilitar la acción de la enzima. En este trabajo se plantea la adición de disolventes miscibles e inmiscibles como alternativas paraincrementar la solubilidad de un HAP modelo tal como el antraceno de cara a su degradación enzimática

Valorization of horse chestnut burs to produce simultaneously valuable compounds under a green integrated biorefinery approach

11 pages, 5 figures, 6 tablesA biorefinery scheme for the valorization of horse chestnut biowastes (a municipal solid waste) into added value bioactive compounds is proposed in this work. The bur fraction of horse chestnut was evaluated as a novel and cheap renewable feedstock to obtain valuable compounds suitable for their use in industrial applications. The integrated valorization scheme comprised an initial hydroethanolic extraction of antioxidant compounds (optimized through surface response methodology), the alkaline delignification of the exhausted solid to obtain a lignin-enriched fraction, and the enzymatic digestibility of the remaining cellulose fraction to produce fermentable sugars. In addition, the structural characterization of the extract by FT-IR and TGA was performed, and the analysis by UPLC-DAD-ESI-MS allowed the tentative identification of eleven antioxidant phenolic compounds. The application of this multiproduct valorization approach led to the production of 13 kg antioxidant extracted compounds, 33.2 kg lignin and 14.5 kg glucose per each 100 kg of horse chestnut burs, which demonstrates the great potential of this residue as a biorefinery substrateThis work was funded by the Ministry of Science, Innovation and Universities (RTI2018-094482-J-I00). G.E., A.M.-M. and T.A.L-C. belong to the Galician Competitive Research Group GRC-ED431C 2017/29 and the CRETUS Strategic Partnership (ED431E 2018/01). These programs are co-funded by FEDER (EU). Beatriz Gullón and Gemma Eibes would like to express their gratitude to the Ministry of Science, Innovation and Universities for financial support (Grant reference RYC2018-026177-I and RYC2018-024846-I). Abel Muñiz-Mouro thanks the Programa de axudas á etapa predoutoral da Xunta de GaliciaPeer reviewe

Formulation of Laccase Nanobiocatalysts Based on Ionic and Covalent Interactions for the Enhanced Oxidation of Phenolic Compounds

Oxidative biocatalysis by laccase arises as a promising alternative in the development of advanced oxidation processes for the removal of xenobiotics. The aim of this work is to develop various types of nanobiocatalysts based on laccase immobilized on different superparamagnetic and non-magnetic nanoparticles to improve the stability of the biocatalysts. Several techniques of enzyme immobilization were evaluated based on ionic exchange and covalent bonding. The highest yields of laccase immobilization were achieved for the covalent laccase nanoconjugates of silica-coated magnetic nanoparticles (2.66 U mg−1 NPs), formed by the covalent attachment of the enzyme between the aldehyde groups of the glutaraldehyde-functionalized nanoparticle and the amino groups of the enzyme. Moreover, its application in the biotransformation of phenol as a model recalcitrant compound was tested at different pH and successfully achieved at pH 6 for 24 h. A sequential batch operation was carried out, with complete recovery of the nanobiocatalyst and minimal deactivation of the enzyme after four cycles of phenol oxidation. The major drawback associated with the use of the nanoparticles relies on the energy consumption required for their production and the use of chemicals, that account for a major contribution in the normalized index of 5.28 × 10−3. The reduction of cyclohexane (used in the synthesis of silica-coated magnetic nanoparticles) led to a significant lower index (3.62 × 10−3); however, the immobilization was negatively affected, which discouraged this alternative