Maja Leitgeb

New findings about the lipase acetylation of nanofibrillated cellulose using acetic anhydride as acyl donor.

The acetylation efficiency of nanofibrillated cellulose (NFC) with acetic anhydride as acetyl donor was studied using lipase from Aspergillus niger in a mixture of dimethyl sulphoxide (DMSO) and phosphate buffer solution at ambient conditions and in supercritical carbon dioxide (scCO2). The chemical acetylation of NFC with comparable ester content was carried out for comparison. The ATR-FTIR, solid-state CP/MAS (13)C NMR and DSC analyses revealed that, besides the enzyme-catalysed acetylation, predominantly appearing at the C-6 position of cellulose hydroxyls, a strong and stable acyl-enzyme intermediate attachment also occurred on the NFC via Maillard reaction. Enzymatic acetylation via attached acyl-enzyme complex on NFC yielded high hydophobicity (contact angle of 84±9°), whereas the chemical acetylation with comparable ester content resulted in a much lower hydrophobic surface with a contact angle of 33±3°. Finally, the adsorption capacity profiles of lysozyme and BSA proteins on native, chemically and enzymatically acetylated NFC as a function of the pH medium were determined.

Different preparation methods and characterization of magnetic maghemite coated with chitosan

The preparation of maghemite (γ-Fe2O3) micro- and nanoparticles coated with chitosan, used as carriers for immobilized enzymes, was investigated. γ-Fe2O3 nanoparticles were synthesized by coprecipitation of Fe2+ and Fe3+ ions in the presence of ammonium. They were coated with chitosan by the microemulsion process, suspension cross-linking technique, and covalent binding of chitosan on the γ-Fe2O3 surface. The methods distinguished the concentration of chitosan, concentration of acetic acid solution, concentration of a cross-linking agent, temperature of synthesis, pH of the medium, and time of synthesis. γ-Fe2O3 micro- and nanoparticles coated with chitosan prepared after three preparation methods were evaluated by scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy analysis, energy dispersive spectrometry, thermogravimetric analysis, differential scanning calorimetry analysis, vibrating sample magnetometry, dynamic light scattering, laser diffraction granulometry, and X-ray diffractometry. These positive attributes demonstrated that these magnetic micro- and nanoparticles coated with chitosan may be used as a promising carrier for further diverse biomedical applications.

NADH oxidation in a microreactor with an oscillating magnetic field

In this study, magnetic nanoparticles (MNPs) of maghemite (γ-Fe2O3) were synthesized and characterized. The method of multifactor experimental design and evolutionary operation (EVOP) was used to optimize immobilization of the alcohol dehydrogenase (ADH) enzyme on MNPs. Optimal operating conditions for the immobilization process were determined (γADH = 0.08 mg/mL, 2% glutaraldehyde for surface activation, t = 28 h), and in such conditions, a specific activity of S.A. = 118 ± 6 U/mg and immobilization efficiency of η = 84.97 ± 3.67% were achieved. Compared to the native enzyme, ADH immobilized on MNPs retained 66.45 ± 3.66% of the initial activity. ADH immobilized on MNPs at optimal conditions was used as a biocatalyst for model reaction—NADH oxidation. NADH oxidation was performed in two different magnetic microreactor configurations: (1) microreactor equipped with permanent square magnets and (2) microreactor equipped with an electromagnet and an oscillating magnetic field that enables magnetic particles movement in the microreactor. In the system with the oscillating magnetic field, equal conversion (X = 100%) was achieved in 2-fold shorter residence time.

NADH oxidation in a microreactor catalysed by ADH immobilised on γ-Fe2O3 nanoparticles

Abstract A new concept of nicotinamide adenine dinucleotide hydrate (NADH) oxidation which combines advantages of the microreactor technology with the advantages of magnetic nanoparticles (MNPs) application is developed. Acetaldehyde was used as a substrate for the NADH regeneration process while the reaction was performed in a batch reactor and in a microreactor using alcohol dehydrogenase (ADH)-loaded MNPs. Three different microreactor systems with MNPs were studied, two with stationary MNPs trapped on the inner surface of microchannel by permanent magnetic field and one where the MNPs actively moved across the channel (movement inside microchannel allowed by an oscillating magnetic field). In a reactor system with an oscillating magnetic field and an actively moving ADH-loaded MNPs 100% NADH conversion was achieved for residence time of just 2 min.

Micro‐ and Nanocarriers for Immobilization of Enzymes

Two types of micro‐ and nanocarriers for immobilization of enzymes for biotechno‐ logical and biomedical applications are described: magnetic nanoparticles and cross‐ linked enzyme aggregates (CLEAs). Nanosized structures with their large surface and smaller size volume ratio, which is dependent on their strong magnetic dipole, give key features that make magnetic nanoparticles useful in many biotechnological and biomedical applications. They are therefore used as carriers to which different active substances can bind. The preparation of the magnetic nanoparticles, possible surface coating methods, and functionalization with different materials are described. Enzyme immobilization methods, such as adsorption, affinity binding, chelation, or metal binding or covalent binding, enable the preparation of efficient and stable enzyme bound to magnetic nanoparticles. Such a product may be used among bioreactor applications for targeted drug delivery in biosensors or bioimaging and magnetic resonance imaging. Preparation of CLEAs, the microsized enzyme structures without a carrier, is described as well. Their main advantage is very simple preparation, where two steps, precipitation of the enzyme and cross‐linking, are joined. A broad spectrum of enzymes for CLEA preparation has been used and many biotechnological reactions are catalyzed. The improvement in CLEA preparation to enhance their stability and operability is also shown.

Enzymatic Reactions in Supercritical Fluids

Supercritical fluids and dense gases are a unique class of non-aqueous media with many features that make their use as solvents for biocatalysis and separation particularly desirable. The advantages of supercritical fluids as solvents fall into four general categories: environmental, process, chemical and health/safety. Other attractive features of supercritical fluids as solvents for biocatalytic processes include their high diffusivities, low toxicity and environmental impact, easy downstream processing and recyclability. Application of dense gases as “green solvents” for biochemical reactions is not yet realized on industrial scale. The reason might be instability and deactivation of enzymes under pressure and temperature.

Use of non-conventional cell disruption method for extraction of proteins from black yeasts

The influence of pressure and treatment time on cells disruption of different black yeasts and on activities of extracted proteins using supercritical carbon dioxide process was studied. The cells of three different black yeasts Phaeotheca triangularis, Trimatostroma salinum, and Wallemia ichthyophaga were exposed to supercritical carbon dioxide (SC CO2) by varying pressure at fixed temperature (35°C). The black yeasts cell walls were disrupted, and the content of the cells was spilled into the liquid medium. The impact of SC CO2 conditions on secretion of enzymes and proteins from black yeast cells suspension was studied. The residual activity of the enzymes cellulase, β-glucosidase, α-amylase, and protease was studied by enzymatic assay. The viability of black yeast cells was determined by measuring the optical density of the cell suspension at 600 nm. The total protein concentration in the suspension was determined on UV–Vis spectrophotometer at 595 nm. The release of intracellular and extracellular products from black yeast cells was achieved. Also, the observation by an environmental scanning electron microscopy shows major morphological changes with SC CO2-treated cells. The advantages of the proposed method are in a simple use, which is also possible for heat-sensitive materials on one hand and on the other hand integration of the extraction of enzymes and their use in biocatalytical reactions.

Chapter 4:Enzyme-based Biomass Catalyzed Reactions in Supercritical CO2

In this work, enzyme-based biomass catalysed reactions in supercritical carbon dioxide (scCO2) are described. As organic solvents are facing upcoming increased environmental concerns and represent an ever-growing class of air pollutants, reduction of them in chemical processes is inevitable. ScCO2 represents a green solvent and an alternative, since it is environmentally neutral and a non-toxic medium. Supercritical fluids (SCFs) are different from organic solvents in having both liquid-like dissolving power and gas-like low viscosities and high diffusivities. Since small changes in pressure or temperature lead to significant changes in density and density-dependant properties, the benefit of using SCFs in different enzymatic reactions and enzyme-based biomass catalysed reactions is widely reported.

Microbial Cellulase Applications in Algal Research

The technical potential of micro- and macroalgae for greenhouse gas abatement has been recognized for many years. Biofuel production from these marine resources, whether using algae for biomass or the potential of some species to produce high-value products, is now an increasing discussion topic. The cell walls of algae consist of a polysaccharide and glycoprotein matrix providing the cells with a formidable defense against its environment. Cellulases are enzymes that can digest the cell wall for the purpose of acquisition of protoplasts, biomass, biofuels, food, and drugs. Effectiveness of the use of cellulases is often limited in their industrial implementation due to their high cost. Using them in an immobilized form enables their repeated use for many cycles without any significant loss of activity and enables reduction of production costs.