Maja Habulin

Compressed gases as alternative enzymatic-reaction solvents: a short review

Abstract Application of supercritical fluids and liquid gases as an alternative reaction medium for enzyme-catalysed reactions is presented. The factors affecting the enzyme stability in supercritical fluids (effect of water activity, effect of pressure and temperature, number of pressurisation–depressurisation steps) and inhibition of enzymes are well documented with experimental results on hydrolases. An overview of our research on various types of HP enzymatic batch-stirred-tank-, continuous-packed-bed- and continuous-membrane reactors to obtain data on productivity and to evaluate the economy of the process is given.

Enzymatic synthesis of oleyl oleate in dense fluids

Esterification between oleic acid and oleyl alcohol, catalyzed by theMucor miehei immobilized lipase in a batch-stirred tank reactor with supercritical carbon dioxide as solvent produced higher reaction rates at supercritical conditions than in the solvent-free system. A continuous fixed-bed reactor was designed based on the results obtained from batch experiments. At 150 bar, 40°C, and with water activity 0.46% w/w, the activity of the enzyme preparation is practically unchanged when CO2 was used as solvent. The addition of small amounts of water increases the conversion rate. The higher conversion also was observed at longer residence time. Whenn-butane was used as reaction medium, a decrease in conversion was observed.

Ionic liquids as (co)solvents for enzymatic reactions

Ionic liquids are low melting point salts that represent an exciting new class of reaction solvents. Many reactions show advantages when carried out in ionic liquids, either with regard to enhanced reaction rates, improved selectivity, or easier reuse of catalysts. To ascertain the influence of ionic liquids on the enzyme activity, three different ionic liquids 1-butyl-3-methylimidazolium chloride ([bmim] [CI]) 1-butyl-3-methylimidazolium hexafluorophosphate ([bmim] [PF6]) and 1-butyl-3-methylimidazolium tetrafluoroborate ([bmim][BF4]) were synthesized and investigated as potential media for the hydrolysis of carboxymethyl cellulose, catalyzed by non-immobilized cellulase from Humicola insolens (Celluzyme 0,7T) and for ester synthesis, catalyzed by immobilized lipase from Rhizomucor miehei (Lipozyme RM IM). Enzyme-catalyzed reactions were performed in a batch stirred reactor at atmospheric pressure. Celluzyme 0,7T showed better activity in hydrophobic ionic liquid ([bmim] [PF6]), as compared to hydrophilic ionic liquid ([bmim] [BF4]). In the case of Lipozyme RM IM, the synthetic activity of the enzyme was strongly reduced by incubating the enzyme in ionic liquids.

Lipase‐catalyzed synthesis of oleyl oleate in pressurized and supercritical solvents

Supercritical and pressurized solvents were used as a biochemical reaction media. As a model system esterification of oleic acid with oleyl alcohol, catalyzed by lipase from Rhizomucor miehei (LipozymeIM) was used. Due to the limitation of the process that may arise from the non-polarity of carbon dioxide, which preferentially dissolves hydrophobic compounds, studies were performed also with other gases (n-butane, n-propane, n-propane:n-butane mixture). The initial reaction rates at 20°C are in n-butane around 5 mmol/(g∗h) and in the mixture n-propane:n-butane around 6 mmol/(g∗h). They are approximately 2–3 times higher than in CO2. At 50°C the reaction rates in n-butane and in n-propane:n-butane mixture are higher in comparison with CO2 only at the pressure, which is subcritical for CO2. The maximal initial reaction rate of 13 mmol/(g∗h) was observed in SC-CO2 at 250 bar and 50°C. The maximal conversion of 87.1% was observed in n-butane at 20°C and 20 bar but the reaction time was 5 h. 86% conversion was reached in only one hour at 50°C and 100 bar in SC-CO2. Lipase-katalysierte Synthese von Oleylolsaure in kompressiblen und superkritischen Losungsmitteln In dieser Arbeit wird die Anwendung kompressibler Losungsmittel (z. B. nahekritische Medien oder Gase) als Reaktionsmedium vorgestellt. Die Veresterung von Olsaure mit Oleylalkohol, katalysiert durch Lipase aus Rhizomucor miehei, wurde als Modellsystem verwendet. Wegen der Unpolaritat des CO2 und der daraus resultierenden geringeren Loslichkeit der hydrophoben Substanzen wurden noch andere kompressible Medien eingesetzt: n-Butan, n-Propan und eine Mischung aus n-Propan:n-Butan. Die Anfangsreaktionsgeschwindigkeit betrug in n-Butan bei 20°C 5 mmol/(g∗h) und in der Gasmischung n-Propan:n-Butan 6 mmol/(g∗h). Das ist ungefahr 2–3mal hoher als bei CO2. Bei 50°C waren die Anfangsreaktionsgeschwindigkeiten in n-Butan und in der Gasmischung n-Propan:n-Butan hoher als bei CO2, was im Bereich der subkritischen Drucke liegt. Die maximale Reaktionsgeschwindigkeit wurde fur SC-CO2 bei 50°C und 250 bar bestimmt. Der maximale Umsatz war 87,1% in n-Butan bei 20°C und 20 bar, jedoch lag die Reaktionszeit bei 5 Stunden. 86% Umsatz konnten in nur einer Stunde bei 50°C und 100 bar in SC-CO2 erzielt werden.

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).

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.

Enzyme-catalyzed reactions in different types of high-pressure enzymatic reactors

The enzyme-catalyzed hydrolysis of carboxy-methyl cellulose (CMC) was performed in three different types of reactors; in a batch stirred-tank reactor (BSTR) operating at atmospheric pressure, in a high-pressure batch stirred-tank reactor (HP BSTR) and in a high-pressure continuous tubular-membrane reactor (HP CTMR). In the high-pressure reactors aqueous SC CO2 was used as the reaction medium. The aim of our research was optimization of the reaction parameters for reaction performance. All the reactions were catalyzed by cellulase from Humicola insolens. Glucose production in the high-pressure batch stirred-tank reactor was faster than in the BSTR at atmospheric pressure. The optimal temperature for the reaction performed in the BSTR at atmospheric pressure was 30°C, while the optimal temperature for the reaction performed in SC CO2 was 32°C. The influence of the application of tubular ceramic membranes in the high-pressure reaction system was studied on the model reaction of CMC hydrolysis at atmospheric pressure and in SC CO2. The reaction was catalyzed by cellulase from Humicola insolens covalently linked to the surface of the ceramic membrane. The hydrolysis of CMC in SC CO2 and at atmospheric pressure was performed for a long time period. The reaction carried out in SC CO2 was more productive than the reaction performed at atmospheric pressure.

Dense Gases as Reaction Media

ABSTRACT Supercritical carbon dioxide (SC-CO 2 ), n-butane and mixture of n-propane:n-butane were used as reaction media for esterification of oleic acid with oleyl alcohol catalyzed by immobilized lipase from Rhizomucor miehei . Syntheses were performed in different reactors: batch stirred tank reactor and packed bed continuous operated reactor at atmospheric pressure and at high pressure. Influence of reaction parameters on initial reaction rates as well as on equilibrium conversion was studied. Results obtained in all types of reactor were compared.