Novel Approach for the Isolation and Immobilization of a Recombinant Transaminase: Applying an Advanced Nanocomposite System.

The increasing application of recombinant enzymes demands not only effective and sustainable fermentation, but also highly efficient downstream processing and further stabilization of the enzymes by immobilization. In this study, a novel approach for the isolation and immobilization of His-tagged transaminase from Chromobacterium violaceum (CvTA) has been developed. A recombinant of CvTA was simultaneously isolated and immobilized by binding on silica nanoparticles (SNPs) with metal affinity linkers and additionally within poly(lactic acid) (PLA) nanofibers. The linker length and the nature of the metal ion significantly affected the enzyme binding efficiency and biocatalytic activity of CvTA-SNPs. The formation of PLA nanofibers by electrospinning enabled rapid embedding of CvTA-SNPs biocatalysts and ensured enhanced stability and activity. The developed advanced immobilization method reduces the time required for enzyme isolation, purification and immobilization by more than fourfold compared to a classical stepwise technique.

Evaluation of biotreatability of ionic liquids in aerobic and anaerobic conditions.

The aim of our study was to set up an approach for reliable biotreatability assessment of ionic liquids (ILs). As a case study, two different ILs were selected: pyridinium-based 1-butyl-3-methylpyridinium dicyanamide ([bmpyr][dca]) and imidazolium-based 1-butyl-3-methylimidazole tetrafluoroborate ([bmim][BF4]). Toxicity in aerobic conditions was determined by measurement of inhibition of oxygen consumption by activated sludge, while their biodegradability was calculated from measurements of oxygen consumption and dissolved organic carbon elimination. For their biotreatability in anaerobic conditions, the method with measurement of biogas production has been employed comparing flocculent and granular sludge. Both ILs were less toxic and more biodegradable in anaerobic conditions. IL [bmpyr][dca] was not toxic to granular sludge up to 742 mg L(-1) and it even has been degraded at this concentration in the presence of easily degradable glucose. Flocculent sludge was completely inhibited at the lower concentration of 318 mg L(-1), but it degraded by 44% at 106 mg L(-1) in the presence of glucose, indicating the appearance of cometabolism. IL [bmim][BF4] was less toxic but also resistant to biodegradation in anaerobic conditions. It degraded via cometabolism 21% at 1,452 mg L(-1). It has been concluded that any assessment of biotreatability of ILs should include parallel determination in aerobic and anaerobic conditions.

Tuning Mechanical Characteristics and Permeability of Alginate Hydrogel by Polyvinyl Alcohol and Deep Eutectic Solvent Addition.

Alginate-based hydrogels are widely utilized for various applications, including enzyme immobilization and the development of drug delivery systems, owing to their advantageous characteristics, such as low toxicity, high availability and cost-effectiveness. However, the broad applicability of alginate hydrogels is hindered by their limited mechanical and chemical stability, as well as their poor permeability to hydrophobic molecules. In this study, we addressed the mechanical properties and chemical resistance of alginate hydrogels in a high-pKa environment by the copolymerization of alginate with polyvinyl alcohol (PVA). The addition of PVA resulted in a threefold improvement in the shear modulus of the copolymeric hydrogel, as well as enhanced chemical resistance to (S)-α-methylbenzylamine, a model molecule with a high pKa value. Furthermore, we addressed the permeability challenge by introducing a betaine-propylene glycol deep eutectic solvent (DES) into the PVA-alginate copolymer. This led to an increased permeability for ethyl 3-oxobutanoate, a model molecule used for bioreduction to chiral alcohols. Moreover, the addition of the DES resulted in a notable improvement of the shear modulus of the resulting hydrogel. This dual effect highlights the role of the DES in achieving the desired improvement of the hydrogel as an immobilization carrier.

Immobilization of His6-tagged amine transaminases in microreactors using functionalized nonwoven nanofiber membranes.

Process intensification is crucial for industrial implementation of biocatalysis and can be achieved by continuous process operation in miniaturized reactors with efficiently immobilized biocatalysts, enabling their long-term use. Due to their extremely large surface-to-volume ratio, nanomaterials are promising supports for enzyme immobilization. In this work, different functionalized nanofibrous nonwoven membranes were embedded in a two-plate microreactor to enable immobilization of hexahistidine (His6)-tagged amine transaminases (ATAs) in flow. A membrane coated with Cu2+ ions gave the best results regarding His6-tagged ATAs immobilization among the membranes tested yielding an immobilization yield of up to 95.3 % for the purified N-His6-ATA-wt enzyme. Moreover, an efficient one-step enzyme immobilization process from overproduced enzyme in Escherichia coli cell lysate was developed and yielded enzyme loads up to 1088 U mL-1. High enzyme loads resulted in up to 80 % yields of acetophenone produced from 40 mM (S)-α-methylbenzylamine in less than 4 min using a continuously operated microreactor. Up to 81 % of the initial activity was maintained in a 5-day continuous microreactor operation with immobilized His6-tagged ATA constructs. The highest turnover number within the indicated time was 7.23·106, which indicates that this immobilization approach using advanced material and reactor system is highly relevant for industrial implementation.

Development of a Microbioreactor for Bacillus subtilis Biofilm Cultivation.

To improve our understanding of Bacillus subtilis growth and biofilm formation under different environmental conditions, two versions of a microfluidic reactor with two channels separated by a polydimethylsiloxane (PDMS) membrane were developed. The gas phase was introduced into the channel above the membrane, and oxygen transfer from the gas phase through the membrane was assessed by measuring the dissolved oxygen concentration in the liquid phase using a miniaturized optical sensor and oxygen-sensitive nanoparticles. B. subtilis biofilm formation was monitored in the growth channels of the microbioreactors, which were designed in two shapes: one with circular extensions and one without. The volumes of these microbioreactors were (17 ± 4) µL for the reactors without extensions and (28 ± 4) µL for those with extensions. The effect of microbioreactor geometry and aeration on B. subtilis biofilm growth was evaluated by digital image analysis. In both microbioreactor geometries, stable B. subtilis biofilm formation was achieved after 72 h of incubation at a growth medium flow rate of 1 µL/min. The amount of oxygen significantly influenced biofilm formation. When the culture was cultivated with a continuous air supply, biofilm surface coverage and biomass concentration were higher than in cultivations without aeration or with a 100% oxygen supply. The channel geometry with circular extensions did not lead to a higher total biomass in the microbioreactor compared to the geometry without extensions.

The Potential of Brewer's Spent Grain in the Circular Bioeconomy: State of the Art and Future Perspectives.

Brewer's spent grain (BSG) accounts for approximately 85% of the total mass of solid by-products in the brewing industry and represents an important secondary raw material of future biorefineries. Currently, the main application of BSG is limited to the feed and food industry. There is a strong need to develop sustainable pretreatment and fractionation processes to obtain BSG hydrolysates that enable efficient biotransformation into biofuels, biomaterials, or biochemicals. This paper aims to provide a comprehensive insight into the availability of BSG, chemical properties, and current and potential applications juxtaposed with the existing and emerging markets of the pyramid of bio-based products in the context of sustainable and circular bioeconomy. An economic evaluation of BSG for the production of highly valuable products is presented in the context of sustainable and circular bioeconomy targeting the market of Central and Eastern European countries (BIOEAST region).

Let the Biocatalyst Flow.

Industrial biocatalysis has been identified as one of the key enabling technologies that, together with the transition to continuous processing, offers prospects for the development of cost-efficient manufacturing with high-quality products and low waste generation. This feature article highlights the role of miniaturized flow reactors with free enzymes and cells in the success of this endeavor with recent examples of their use in single or multiphase reactions. Microfluidics-based droplets enable ultrahigh-throughput screening and rapid biocatalytic process development. The use of unique microreactor configurations ensures highly efficient contacting of multiphase systems, resulting in process intensification and avoiding problems encountered in conventional batch processing. Further integration of downstream units offers the possibility of biocatalyst recycling, contributing to the cost-efficiency of the process. The use of environmentally friendly solvents supports effective reaction engineering, and thus paves the way for these highly selective catalysts to drive sustainable production.

Hydrogel-Based Enzyme and Cofactor Co-Immobilization for Efficient Continuous Transamination in a Microbioreactor.

A microbioreactor was developed in which selected amine transaminase was immobilized together with the cofactor pyridoxal phosphate (PLP) to allow efficient continuous transamination. The enzyme and cofactor were retained in a porous copolymeric hydrogel matrix formed in a two-plate microreactor with an immobilization efficiency of over 97%. After 10 days of continuous operation, 92% of the initial productivity was retained and no leaching of PLP or enzyme from the hydrogel was observed. The microbioreactor with co-immobilized cofactor showed similar performance with and without the addition of exogenous PLP, suggesting that the addition of PLP is not required during the process. The space-time yield of the microbioreactor was 19.91 g L-1 h-1, while the highest achieved biocatalyst productivity was 5.4 mg mgenzyme -1 h-1. The immobilized enzyme also showed better stability over a wider pH and temperature range than the free enzyme. Considering the time and cost efficiency of the immobilization process and the possibility of capacity expansion, such a system is of great potential for industrial application.

Trametes versicolor in lignocellulose-based bioeconomy: State of the art, challenges and opportunities.

Although Trametes versicolor is one of the most investigated white-rot fungi, the industrial application of this fungus and its metabolites is still far from reaching its full potential. This review aims to highlight the opportunities and challenges for the industrial use of T. versicolor according to the principles of circular bioeconomy. The use of this fungus can contribute significantly to the success of efforts to valorize lignocellulosic waste biomass and industrial lignocellulosic byproducts. Various techniques of T. versicolor cultivation for enzyme production, food and feed production, wastewater treatment, and biofuel production are listed and critically evaluated, highlighting bottlenecks and future perspectives. Applications of T. versicolor crude laccase extracts in wastewater treatment, removal of lignin from lignocellulose, and in various biotransformations are analyzed separately.

Immobilization of yeast cells within microchannels of different materials.

Saccharomyces cerevisiae was successfully immobilized on the inner wall surface of channels of submillimeter diameter, which can be further used for the development of a highly productive continuous biotransformation process within a microfluidic device. Covalent bonding by means of 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde was used for immobilization of cells to microchannels made of glass, polystyrene (PS), polytetrafluoroethylene (PTFE), perfluoroalkoxy (PFA) and fluorinated ethylene propylene (FEP). All tested materials were successfully functionalized with H2SO4 to promote silanization. The effect of reaction time with acid on immobilization efficiency was studied for polymer materials. This is the first report on cell immobilization onto PTFE, FEP and PFA surface, which enables to develop a microfluidic device with surface bound biocatalyst from low cost and disposable materials.

Oxidation of Coniferyl Alcohol Catalyzed by Laccases from Trametes versicolor.

Oxidation of coniferyl alcohol catalyzed by commercial laccase and crude laccase produced during the submerged cultivation of Trametes versicolor MZKI G-99 in a medium containing the waste from paper industry was investigated. pH of 6.6 and temperature of 35 °C was found to be optimal for coniferyl alcohol oxidation catalyzed by commercial laccase. Based on the initial reaction rate measurements, apparent Michaelis-Menten kinetic parameters for commercial laccase were determined in an aqueous media (Vm = 4.387 U mg-1, Km = 0.025 mmol dm-3), as well as in 1:1 (v/v) methanol: phosphate buffer mixture (Vm = 0.979 U mg-1, Km = 0.019 mmol dm-3). Inhibition of substrate was found for crude laccase and the following apparent kinetic parameters Vm = 9.272 U mg-1, Km = 0.045 mmol dm-3 and Ki = 0.002 mmol dm-3 were estimated. Mathematical model of batch process, which includes double-substrate Michaelis-Menten kinetics with oxygen as the second substrate and mass balances, has been developed and validated in experiments with or without additional aeration. 100 % conversions of up to 0.8 mmol dm-3 of coniferyl alcohol in batch experiment due to the high operational stability of enzymes was realized with both laccases.

Covalent Immobilization of Microbial Cells on Microchannel Surfaces.

Microfluidic devices with integrated biological material have found many applications in analytics (e.g., protein and DNA analysis), biochemistry (e.g., PCR), and medical diagnostics (e.g., ELISA test). Recently they are also considered as promising tools for bioprocess development and intensification. In order to enable long-term biocatalyst use and to facilitate its separation from the product, immobilization within the microreactor is often preferred over the use of free enzymes or cells. Surface immobilization is frequently selected due to the very high surface-to-volume ratio of microfluidic devices that offers the possibility for high biocatalyst load and at the same time good biocatalyst accessibility. Moreover, such reactor design prevents the increase in backpressure, often encountered in packed-bed or monolithic microreactors.Microbial cells are beneficial over the isolated enzymes in many biotransformations, especially in multistep syntheses and in cofactor-dependent reactions. Their immobilization within microreactors, especially made from disposable polymers, is of a big interest for analytical and synthetic applications.This chapter describes procedure for immobilization of eukaryotic and prokaryotic cells onto inner surfaces of microreactors made from various polymeric materials and glass. Cells could be immobilized in high densities and remain stably attached over several days of continuous microreactor operation.

Development of a Microfluidic Platform for R-Phycoerythrin Purification Using an Aqueous Micellar Two-Phase System.

Temperature-dependent aqueous micellar two-phase systems (AMTPSs) have recently been gaining attention in the isolation of high-added-value biomolecules from their natural sources. Despite their sustainability, aqueous two-phase systems, and particularly AMTPSs, have not been extensively applied in the industry, which might be changed by applying process integration and continuous manufacturing. Here, we report for the first time on an integrated microfluidic platform for fast and low-material-consuming development of continuous protein purification using an AMTPS. A system comprised of a microchannel incubated at high temperature, enabling instantaneous triggering of a two-phase system formation, and a microsettler, allowing complete phase separation at the outlets, is reported here. The separation of phycobiliproteins and particularly the purification of R-phycoerythrin from the contaminant proteins present in the aqueous crude extract obtained from fresh cells of Gracilaria gracilis were thereby achieved. The results from the developed microfluidic system revealed that the fractionation performance was maintained while reducing the processing time more than 20-fold when compared with the conventional lab-scale batch process. Furthermore, the integration of a miniaturized ultrafiltration module resulted in the complete removal of the surfactant from the bottom phase containing R-phycoerythrin, as well as in nearly twofold target protein concentration. The process setup successfully exploits the benefits of process intensification along with the integration of various downstream processes. Further transfer to a meso-scale integrated system would make such a system appropriate for the separation and purification of biomolecules with high commercial interest.

The Promises and the Challenges of Biotransformations in Microflow.

The challenges of transition toward the postpetroleum world shed light on the biocatalysis as the most sustainable way for the valorization of biobased raw materials. However, its industrial exploitation strongly relies on integration with innovative technologies such as microscale processing. Microflow devices remarkably accelerate biocatalyst screening and engineering, as well as evaluation of process parameters, and intensify biocatalytic processes in multiphase systems. The inherent feature of microfluidic devices to operate in a continuous mode brings additional interest for their use in chemoenzymatic cascade systems and in connection with the downstream processing units. Further steps toward automation and analytics integration, as well as computer-assisted process development, will significantly affect the industrial implementation of biocatalysis and fulfill the promises of the bioeconomy. This review provides an overview of recent examples on implementation of microfluidic devices into various stages of biocatalytic process development comprising ultrahigh-throughput biocatalyst screening, highly efficient biocatalytic process design including specific immobilization techniques for long-term biocatalyst use, integration with other (bio)chemical steps, and/or downstream processing.

Copolymeric Hydrogel-Based Immobilization of Yeast Cells for Continuous Biotransformation of Fumaric Acid in a Microreactor.

Although enzymatic microbioreactors have recently gained lots of attention, reports on the use of whole cells as biocatalysts in microreactors have been rather modest. In this work, an efficient microreactor with permeabilized Saccharomyces cerevisiae cells was developed and used for continuous biotransformation of fumaric into industrially relevant L-malic acid. The immobilization of yeast cells was achieved by entrapment in a porous structure of various hydrogels. Copolymers based on different ratios of sodium alginate (SA) and polyvinyl alcohol (PVA) were used for hydrogel formation, while calcium chloride and boric or phenylboronic acid were tested as crosslinking agents for SA and PVA, respectively. The influence of hydrogel composition on physico-chemical properties of hydrogels prepared in the form of thin films was evaluated. Immobilization of permeabilized S. cerevisiae cells in the selected copolymeric hydrogel resulted in up to 72% retained fumarase activity. The continuous biotransformation process using two layers of hydrogels integrated into a two-plate microreactor revealed high space time yield of 2.86 g/(L·h) while no activity loss was recorded during 7 days of continuous operation.

Development of microreactors with surface-immobilized biocatalysts for continuous transamination.

The industrial importance of optically pure compounds has thrown a spotlight on ω-transaminases that have shown a high potential for the synthesis of bioactive compounds with a chiral amine moiety. The implementation of biocatalysts in industrial processes relies strongly on fast and cost effective process development, including selection of a biocatalyst form and the strategy for its immobilization. Here, microscale reactors with selected surface-immobilized amine-transaminase (ATA) in various forms are described as platforms for high-throughput process development. Wild type ATA (ATA-wt) from a crude cell extract, as well as Escherichia coli cells intracellularly overexpressing the enzyme, were immobilized on the surfaces of meander microchannels of disposable plastics by means of reactor surface silanization and glutaraldehyde bonding. In addition, a silicon/glass microchannel reactor was used for immobilization of an ATA-wt, genetically engineered to contain a silica-binding module (SBM) at the N-terminus (N-SBM-ATA-wt), leading to immobilization on the non-modified inner microchannel surface. Microreactors with surface-immobilized biocatalysts were coupled with a quenching system and at-line HPLC analytics and evaluated based on continuous biotransformation, yielding acetophenone and l-alanine. E. coli cells and N-SBM-ATA-wt were efficiently immobilized and yielded a volumetric productivity of up to 14.42 g L-1 h-1, while ATA-wt small load resulted in two orders of magnitude lower productivity. The miniaturized reactors further enabled in-operando characterization of biocatalyst stability, crucial for successful transfer to a production scale.

Steroid extraction in a microchannel system--mathematical modelling and experiments.

The continuous ethyl acetate extraction of progesterone and 11alpha-hydroxyprogesterone, a reactant and the product of the biotransformation step involved in corticosteroid production, was studied in a microchannel at different flow velocities. In addition, non-steady state batch extraction without mixing was performed and modelled in order to verify the theoretically predicted parameters. In order to analyze experimental data and to forecast microreactor performance, a three-dimensional mathematical model with convection and diffusion terms was developed considering the velocity profile for laminar flow of two parallel phases in a microchannel at steady-state conditions. For the numerical solution of a complex equation system, non-equidistant finite differences were used. Very good agreement between model calculations and experimental data was achieved without any fitting procedure. Due to the efficient phase separation and high extraction yields obtained, the micro scale extraction units were found to be a promising tool for the development of an integrated system of 11alpha-hydroxylation of progesterone by Rhizopus nigricans in the form of pellets.

Microscale technology and biocatalytic processes: opportunities and challenges for synthesis.

Despite the expanding presence of microscale technology in chemical synthesis and energy production as well as in biomedical devices and analytical and diagnostic tools, its potential in biocatalytic processes for pharmaceutical and fine chemicals, as well as related industries, has not yet been fully exploited. The aim of this review is to shed light on the strategic advantages of this promising technology for the development and realization of biocatalytic processes and subsequent product recovery steps, demonstrated with examples from the literature. Constraints, opportunities, and the future outlook for the implementation of these key green engineering methods and the role of supporting tools such as mathematical models to establish sustainable production processes are discussed.

Optimization of laccase production by Trametes versicolor cultivated on industrial waste.

Laccases are very interesting biocatalysts for several industrial applications. Its production by different white-rot fungi can be stimulated by a variety of inducing substrates, and the use of lignocellulosic wastes or industrial by-products is one of the possible approaches to reduce production costs. In this work, various industrial wastes were tested for laccase production by Trametes versicolor MZKI G-99. Solid waste from chemomechanical treatment facility of a paper manufacturing plant showed the highest potential for laccase production. Enzyme production during submerged cultivation of T. versicolor on the chosen industrial waste has been further improved by medium optimization using genetic algorithm. Concentrations of five components in the medium were optimized within 60 shake-flasks experiments, where the highest laccase activity of 2,378 U dm(-3) was achieved. Waste from the paper industry containing microparticles of CaCO(3) was found to stimulate the formation of freely dispersed mycelium and laccase production during submerged cultivation of T. versicolor. It was proven to be a safe and inexpensive substrate for commercial production of laccase and might be more widely applicable for metabolite production by filamentous fungi.

Continuous steroid biotransformations in microchannel reactors.

The use of microchannel reactor based technologies within the scope of bioprocesses as process intensification and production platforms is gaining momentum. Such trend can be ascribed a particular set of characteristics of microchannel reactors, namely the enhanced mass and heat transfer, combined with easier handling and smaller volumes required, as compared to traditional reactors. In the present work, a continuous production process of 4-cholesten-3-one by the enzymatic oxidation of cholesterol without the formation of any by-product was assessed. The production was carried out within Y-shaped microchannel reactors in an aqueous-organic two-phase system. Substrate was delivered from the organic phase to aqueous phase containing cholesterol oxidase and the product formed partitions back to the organic phase. The aqueous phase was then forced through a plug-flow reactor, containing immobilized catalase. This step aimed at the reduction of hydrogen peroxide formed as a by-product during cholesterol oxidation, to avoid cholesterol oxidase deactivation due to said by-product. This setup was compared with traditional reactors and modes of operation. The results showed that microchannel reactor geometry outperformed traditional stirred tank and plug-flow reactors reaching similar conversion yields at reduced residence time. Coupling the plug-flow reactor containing catalase enabled aqueous phase reuse with maintenance of 30% catalytic activity of cholesterol oxidase while eliminating hydrogen peroxide. A final production of 36 m of cholestenone was reached after 300 hours of operation.