Mateja Primožič

Enzymatic activity of L-amino acid oxidase from snake venom Crotalus adamanteus in supercritical CO2

L-amino acid oxidase (L-AAO) from snake venom Crotalus adamanteus was successfully tested as a catalyst in supercritical CO2 (SC-CO2). The enzyme activity was measured before and after exposure to supercritical conditions (40°C, 110 bar). It was found that L-AAO activity slightly increased after SC-CO2 exposure by up to 15%. L-AAO was more stable in supercritical CO2 than in phosphate buffer under atmospheric pressure, as well as in the enzyme membrane reactor (EMR) experiment. 3,4-Dihydroxyphenyl-L-alanine (L-DOPA) oxidation was performed in a batch reactor made of stainless steel that could withstand the pressures of SC-CO2, in which L-amino acid oxidase from C. adamanteus was able to catalyze the reaction of oxidative deamination of L-DOPA in SC-CO2. For the comparison L-DOPA oxidation was performed in the EMR at 40°C and pressure of 2.5 bar. Productivity expressed as mmol-s of converted L-DOPA after 3 h per change of enzyme activity after 3 h was the highest in SC-CO2 (1.474 mmol U−1), where catalase was present, and the lowest in the EMR (0.457 mmol U−1).

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.

Application of Ionic Liquids in Biocatalysis

Biotransformations have been of enormous economic and social importance throughout the history of mankind (Liese et al., 2000). Biocatalysis may be the most efficient way of producing fine chemicals. Today, several chemicals like pharmaceuticals, amino acids, saccharides and polysaccharides, esters and vitamins are produced by enzymatic biotransformations on industrial scale (Liese et al., 2000). The production of fine chemicals results in output of considerable volume of waste. Most of wastes are solvents such as water, volatile organic compounds (VOCs) etc. Solvents comprise 2/3 of all industrial emissions and 1/3 of all VOC emissions nationwide. These emissions have been linked to a host of negative effects (global climate change, pollution of air, human illness etc.) (Brennecke & Maginn, 2001). In recent years, green chemistry is become a growing area of research. Therefore the search for new environmental friendly and benign solvents and catalysts which can be easily recycled or reused is of significant interest. The ideal solvent should be chemically and physically stabile, recyclable, and reusable, should have a very low volatility, should allow selective and rapid transformations and should be easy to handle. For the biocatalysis, there are five main “green” solvent systems: supercritical fluids (SCFs), fluorinated solvents, ionic liquids (ILs), water, and solvent free reactions (Hobbs & Thomas, 2007). Enzymatic reactions could be performed under preferred conditions with minimized yield of the undesired by-products. Meanwhile, low yields, selectivity, and poor solubility of substrates in aqueous medium may require the enzymatic reactions to be carried out in non-aqueous medium (Sureshkumar & Lee, 2009). SCF is any substance at a temperature and pressure above its critical point. Close to the critical point, small changes in pressure or temperature result in large changes in density, allowing many properties of a SCF to be “fine-tuned” (Jessop & Leitner, 1999). There are several advantages using the SCFs as solvents in chemical synthesis, where all are based on unique thermo-physical properties of SCFs for their mixtures with reactants. The main advantage of biocatalysis in SCFs is the tunability of the properties of the solvent by changes in the pressure and/or the temperature. The application of SCFs enables also design of integrated reaction and separation processes. In mass transfer limited processes the reaction rate can be increased if SCFs are applied due to higher diffusivity and to reduce viscosity of reaction system. SCFs display unique substrate specificity at relative mild reaction conditions.

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.

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.