3D Printed Polylactic Acid Well-Plate for Multi-enzyme Immobilization.

3D printing is lately utilized in biological sciences under the scope to develop customized scaffolds that will host biomolecules, either whole cells or parts of them, like enzymes. In the present work, we present a protocol to modify the surface of 3D printed polylactic acid (PLA) well-plates with the aim to co-immobilize multiple enzymes that will perform cascade reactions. Detailed steps to design and print the final models are described. The developed protocol for surface modification is based on coating with chitosan biopolymer and covalent immobilization of the enzymes ß-glucosidase, glucose oxidase, and peroxidase via glutaraldehyde cross-linking. Enzymatic activity measurements indicative of the catalytic performance of the system are also presented.

Development of 3D Printed Enzymatic Microreactors for Lipase-Catalyzed Reactions in Deep Eutectic Solvent-Based Media.

In this study, 3D printing technology was exploited for the development of immobilized enzyme microreactors that could be used for biocatalytic processes in Deep Eutectic Solvent (DES)-based media. 3D-printed polylactic acid (PLA) microwell plates or tubular microfluidic reactors were modified with polyethylenimine (PEI) and lipase from Candida antarctica (CALB) was covalently immobilized in the interior of each structure. DESs were found to have a negligible effect on the activity and stability of CALB, and the system proved highly stable and reusable in the presence of DESs for the hydrolysis of p-nitrophenyl butyrate (p-NPB). A kinetic study under flow conditions revealed an enhancement of substrate accessibility in the presence of Betaine: Glycerol (Bet:Gly) DES, while the system was not severely affected by diffusion limitations. Incubation of microreactors in 100% Bet:Gly preserved the enzyme activity by 53% for 30 days of storage at 60 °C, while the buffer-stored sample had already been deactivated. The microfluidic enzyme reactor was efficiently used for the trans-esterification of ethyl ferulate (EF) with glycerol towards the production of glyceryl ferulate (GF), known for its antioxidant potential. The biocatalytic process under continuous flow conditions exhibited 23 times higher productivity than the batch reaction system. This study featured an effective and robust biocatalytic system with immobilized lipase that can be used both in hydrolytic and synthetic applications, while further optimization is expected to upgrade the microreactor system performance.

3D printed PLA enzyme microreactors: Characterization and application for the modification of bioactive compounds.

Process sustainability of biocatalytic processes is significantly empowered with the use of continuous-flow technologies that offer high productivity, minimal wastes and low volumetric consumption. Combining microreactor design with 3D printing technology can broaden the engineering potentials. This work proposes a protocol to modify the surface of 3D-printed PLA scaffolds, based on chitosan deposition. Mimicking the concept of microplates, multi-well plates were designed to facilitate parameter testing. Immobilization of laccase from Trametes versicolor was successfully performed, while chitosan and cross-linker concentration and incubation time were optimized. Τhe developed protocol was applied for the continuous flow bioconversion of hydroxyyrosol, yielding a TTN of 438.6 × 103 for a total of 10 h continuous use. Also, a peristaltic flow pattern seemed to favor the system performance, reaching 95% bioconversion efficiency in a total of 1 h reaction time. The potential of the developed system was further evaluated for the biotransformation of different biophenols from dietary sources, proving the efficiency of the system as a versatile biotechnological tool.

Trends in the development of innovative nanobiocatalysts and their application in biocatalytic transformations.

The ever-growing demand for cost-effective and innocuous biocatalytic transformations has prompted the rational design and development of robust biocatalytic tools. Enzyme immobilization technology lies in the formation of cooperative interactions between the tailored surface of the support and the enzyme of choice, which result in the fabrication of tremendous biocatalytic tools with desirable properties, complying with the current demands even on an industrial level. Different nanoscale materials (organic, inorganic, and green) have attracted great attention as immobilization matrices for single or multi-enzymatic systems. Aiming to unveil the potentialities of nanobiocatalytic systems, we present distinct immobilization strategies and give a thorough insight into the effect of nanosupports specific properties on the biocatalysts' structure and catalytic performance. We also highlight the development of nanobiocatalysts for their incorporation in cascade enzymatic processes and various types of batch and continuous-flow reactor systems. Remarkable emphasis is given on the application of such nanobiocatalytic tools in several biocatalytic transformations including bioremediation processes, biofuel production, and synthesis of bioactive compounds and fine chemicals for the food and pharmaceutical industry.

Multienzymatic Nanoassemblies: Recent Progress and Applications.

Biotechnological research has turned to multienzymatic nanoassemblies as a promising concept to host multiple applications. Here, we consider important aspects around the development and optimization of such biocatalytic systems and present current advances in utilizing bi- and multienzymatic cascade reactions in diverse fields, including ultrasensitive biosensing, development of pharmaceuticals, and conversion of natural biopolymers to valuable products, highlighting their future potential in the chemical, biotechnological, and pharmaceutical industries. Diverse co-immobilization techniques and different parameters affecting the performance of multienzymatic cascade reactions are discussed. Continuous flow processes incorporating multienzymatic nanoassemblies in different reactor configurations are also presented. This technology provides an arsenal of tools for the development of innovative and effective multienzymatic systems offering new possibilities for biocatalysts applications.

Enzymatic Conversion of Oleuropein to Hydroxytyrosol Using Immobilized ß-Glucosidase on Porous Carbon Cuboids.

In the present study, we developed novel ß-glucosidase-based nano-biocatalysts for the bioconversion of oleuropein to hydroxytyrosol. Using non-covalent or covalent immobilization approaches, ß-glucosidases from almonds and Thermotoga maritima were attached for the first time on oxidized and non-oxidized porous carbon cuboids (PCC). Various methods were used for the characterization of the bio-nanoconjugates, such as Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and fluorescence spectroscopy. The oxidation state of the nanο-support and the immobilization procedure play a key role for the immobilization efficiency or the catalytic activity of the immobilized ß-glucosidases. The nano-biocatalysts were successfully used for the hydrolysis of oleuropein, which leads to the formation of its bioactive derivative, hydroxytyrosol (up to 2.4 g L-1), which is a phenolic compound with numerous health benefits. The bio-nanoconjugates exhibited high thermal and operational stability (up to 240 hours of repeated use), which indicated that they are efficient tools for various bio-transformations.

Stabilization of Laccase Through Immobilization on Functionalized GO-Derivatives.

This chapter deals with the use of functionalized derivatives of graphene oxide as nanoscaffolds for the immobilization and stabilization of laccase from Trametes versicolor. Covalent and noncovalent immobilization approaches are described, while a novel method for the development of laccase-based multilayer nanoassemblies is also presented. Various biochemical, spectroscopic, and microscopic techniques were applied to characterize the nanobiocatalytic systems in respect to their microstructure and catalytic performance. Laccase-GO nanosystems were characterized with FTIR spectroscopy in order to confirm the functionalization of the nanomaterials, as well as to interpret the nanomaterial-enzyme interactions, while the multilayer structure of laccase-based multilayer nanoassemblies was confirmed by atomic force microscopy. The nanobiocatalytic systems presented here demonstrated exceptional stability and reusability compared with the free enzyme form, leading to robust biocatalytic systems appropriate for various applications of industrial interest.