No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Enzyme-Catalyzed Interesterification of Triglycerides in Supercritical Carbon Dioxide
Abstract
The interesterification of trilaurin and myristic acid, catalyzed by a 1,3-specific lipase from Rhizopus arrhizus, has been investigated in supercritical carbon dioxide. Experimental data have been obtained from reactions conducted in a continuous-flow packed-bed reactor containing lipase covalently attached to glass beads. The reaction rate is not influenced by mass-transfer limitations over the range of flow rates studied, and lipase retains full activity at 1400 psi and 35-degrees-C for up to 80 h. The carbon dioxide water content does not affect the intrinsic activity of the enzyme, but a higher water concentration causes a greater degree of unwanted hydrolysis. The selectivity of the reaction for interesterification over hydrolysis improves at higher pressures as the extent of hydrolysis reaction is reduced. The activity and stability of lipase in supercritical carbon dioxide are similar to those in organic liquid solvents.
... Jackson et al. (1997) applied the flow of oil solution in CO 2 through the bed of immobilised enzyme also to conduct randomization of high stearate soybean oil and palm olein. The kinetics of oil reaction in a continuous packed bed reactor was studied by Miller et al. (1991). Interesterification of trilaurin (representing the oil) and myristic acid was catalysed by immobilised lipase from Rhizopus arrhizus. ...
... where conversion ı in the range 0-1 is the measure of substrate depletion and is the residence time in the reactor. For example, for Michaelis-Menten one-substrate kinetics the relationship between the reaction rate parameters, residence time, conversion and initial substrate concentration is ( Miller et al., 1991) ...
... When the specific reaction rate measured at 15 MPa in series A-F was plotted in logarithmic scale against superficial velocity ( Fig. 4), the straight lines fitted to the points of individual series were shifted vertically because the activity in different enzyme fillings was not identical but the slope of the lines was almost equal to 0.83, the value of exponent in Eq. (5) for mass transfer rate. Thus, the reaction was controlled by mass transfer, contrary to the work of Miller et al. (1991) where superficial velocities 0.7-2.4 mm s −1 were higher than in this work (0.1-0.7 mm s −1 ) and, on the contrary, the observed maximum reaction rate was higher in this work. ...
Blackcurrant seed oil is rich in linoleic and linolenic acids. Selective enzyme-catalysed oil hydrolysis was studied with aim to obtain different levels of α- and/or γ-linolenic acid in the mixture of liberated fatty acids and in the fraction of di- and monoacylglycerols, making them suitable for special dietary needs. The oil was dissolved in supercritical carbon dioxide flowing through a packed bed reactor (temperature 40°C, pressure 15–28MPa, and superficial velocity 0.1–0.7mms−1) with Lipozyme®, a 1,3-specific lipase from Mucor miehei immobilised on a macroporous ion-exchange resin. The enzyme activity was stable as long as water precipitation in the reactor was prevented. The reaction was found to be controlled by both Michaelis–Menten kinetics and mass transfer. The maximum rate of fatty acids liberation per unit amount of enzyme, 2.6×10−3mols−1kg−1, was achieved at the maximum flow velocity and pressure. Compared to oil, the liberated fatty acids contained more α-linolenic, palmitic and stearic acids, while di- and monoacylglycerols contained increased levels of γ-linolenic and stearidonic acids.
... The model parameter fitting to experimental data however led to mass transfer coefficients which were too high to affect the simulated results. This indicates that the mass transfer between bulk fluid and pores of a clean macroporous enzyme was much faster than the reaction and did not affect the process kinetics, similarly like in the study of Miller et al. [26]. As shown above, a significant local mass transfer resistance between pores and bulk can exist but it cannot be expressed by a single mass transfer coefficient because it is related to the retention of a part of reaction products by enzyme bed, which slowly develops and is both space-and time dependent. ...
Research on enzyme-catalysed reactions of oils in supercritical CO2 (scCO2) aims at solvent-free natural products enriched in bioactive substances. Easy tailoring of scCO2 properties and low mass transfer limitations belong to advantages of the method, which perspective of large-scale application depends on enzyme stability in scCO2. Mathematical model for immobilized lipase-catalysed vegetable oil hydrolysis in a continuous-flow reactor involves a slow reversible decrease of enzyme activity caused by glycerol retention in the enzyme bed. Reaction kinetics is modelled as pseudo-first-order and pseudo-second-order reversible hydrolysis of tri-, di-, and monoglycerides. Reaction rate constants and parameters of glycerol adsorption were estimated by fitting the model to previously published experimental data. Conditions of hydrolysis catalysed by Lipozyme TL IM were pressure 10-30 MPa, temperature 30-50 °C, and scCO2 flow rate-to-enzyme load ratio 2-120 g·g⁻¹min⁻¹. The rate controlling step was hydrolysis of triglycerides with pressure and temperature dependent kinetic constant 100-230 min⁻¹.
... Beyond the reaction types, the ultimate enzyme activity is acquirable at a certain amount of water. For instance, it is reported that the maximum efficiency of the interesterification of myristic acid with trilaurin, catalyzed by lipase, was obtained at a lower amount of water mixed with SCF owing to the reduction in trilaurin solubility with increasing the water content [16,48]. On the contrary, it is demonstrated the ultimate enzyme activity for the esterification of myristic acid is achievable at an significant amount of water as co-solvent [16]. ...
This chapter aims to give some insights into supercritical fluid (SCF) applications in reaction systems in view of chemical engineering applications. In chemical reactions, the reaction medium is usually a fluid solvent that must have specific characteristics that can be regulated through temperature and pressure of the system to above its critical point. The supercritical technology can favorably modify the solvent properties as required for reactions. Some applications of SCFs and specifically supercritical water (SCW) are discussed in this chapter including waste destruction, biomass processing, and chemical synthesis. Furthermore, important factors influencing SCF-biocatalytic reaction systems (enzyme-catalyzed reactions) including the effect of water content on enzyme operation in the presence of SCFs, the effect of operation conditions, and the effect of solvent characteristics are also elaborated. A discussion on the challenges and limitations of SCFs for industrial applications is also provided.
... Although for most lipases actions a certain amount of water is essential in initiating the reaction, the control of the water quantity is crucial. In transesterification, excess water may result in undesired reactions, such as triglycerides hydrolysis (Chowdary and Prapulla, 2002;Leitner and Jessop, 2014;Macrae, 1983;Miller et al., 1991). Carrying out transesterification reactions in a solvent-free medium has other drawbacks besides mass transesterifications. ...
... Enzymatic transesterification have been carried out in supercritical fluids (SCF) since many enzymes are active and stable in that media. Miller et al. conducted the interesterification of trilaurin and myristic acid, catalyzed by a lipase from Rhizopus arrhizus, in a continuous-flow packed-bed reactor containing lipase covalently attached to glass beads [111]. Habulin et al. studied the lipase-catalyzed synthesis of oleic acid esters with various primary alcohols. ...
... Changes in the enzyme efficiency at two different pressures (24.0 and 38.0 MPa) during a continuous-flow hydrolysis of canola oil in SCCO 2 are shown in Fig. 3. Significant changes in the enzyme efficiency were observed due to the change in pressure. Although, according to Miller et al. [20], pressure at the levels of this study does not have significant effect on the enzyme activity, certain changes in enzyme efficiency can be attributed to the changes in the medium properties such as phase behavior, diffusion properties and therefore substrate concentrations on the enzyme sites. However, when a continuous drop in the enzyme efficiency is observed at given operational conditions, then it has to be related to possible changes in the enzyme behavior due to a change in its (micro)structure. ...
Enzyme efficiency was investigated in the lipase-catalyzed hydrolysis of canola oil in supercritical carbon dioxide (SCCO(2)). Immobilized lipase from Mucor miehie (Lipozyme IM) was used as the catalyst and the results showed that enzyme efficiency dropped at high pressures indicating a possible change in enzyme microstructure. Therefore, scanning electron microscopy (SEM) was used as a supplementary tool to investigate microstructural stability of the enzyme under supercritical conditions. SEM images of the treated enzymes did not demonstrate any apparent structural changes with a change in pressure (24.0 and 38.0 MPa), enzyme load of the reactor (1.0 and 5.0 g), CO(2) flow rate (0.5 and 3.9 L/min) and the oil content (extracted from 3.0 and 15.0 g canola flakes) of SCCO(2). However, a change at the molecular level is a possibility, which requires further investigation.
... The authors attributed this effect to a higher adsorption of the synthesised ester on the enzyme bed as the solvent capacity of scCO 2 decreases with decreasing pressure. Furthermore, Miller et al. [318,319] reported an increase of reaction rate with pressure. They studied the interesterification of trilaurin and myristic acid in scCO 2 saturated with the substrates at pressures in the range from 80 bar to 110 bar. ...
... In the past decade, a number of studies on enzyme catalysis in supercritical fluids (SCF) have been carried out since many enzymes are active and stable in SCFs. In particular, lipases have been widely used to catalyze a variety of reactions in SC-CO 2 . 1 Miller et al. 2 conducted the interesterification of trilaurin and myristic acid, catalyzed by a 1,3-specific lipase from Rhizopus arrhizus, in a continuous-flow packed-bed reactor containing lipase covalently attached to glass beads. The selectivity of the reaction for interesterification over hydrolysis improved at higher pressures as the extent of hydrolysis reaction was reduced. ...
On-line extraction−reaction was carried out in supercritical carbon dioxide (SC-CO2) in the temperature range from 308 to 328 K and the pressure range from 24 to 35 MPa. Fatty acid ethyl esters were synthesized from extracted canola oil with ethanol by using the immobilized lipase (Lipozyme IM). The effects of ethanol amount, enzyme load, and operational conditions on the amount of fatty acid esters (FAE), free fatty acids (FFA), and mono-, di-, and triglycerides (MG, DG, and TG, respectively) present in the product were investigated. The results show that it is possible to synthesize fatty acid ethyl esters efficiently at high temperature. Finally, a new process operating at different extractor and reactor temperatures was proposed.
... The interesterification of trilaurin and myristic acid, catalyzed by a 1,3-specific lipase from Rhizopus arrhizus, was investigated in supercritical carbon dioxide using a continuous-flow packed-bed reactor containing lipase covalently attached to glass beads (Miller et al., 1991). The reaction rate was not influenced by mass transfer limitations over the range of flow rates studied, and lipase retained full activity at 1400 psi and 35°C for up to 80 hr. ...
Lipids in biological matter are mostly triacylglycerols (TAG). Lipolytic enzymes, primarily lipases, are indispensable for bioconversion of such lipids from one organism to another and within the organisms. In addition to their biological significance, lipases are very important in the field of food technology, nutritional and pharmaceutical sciences, chemical and detergent industries, and clinical medicine because of their ability to catalyze various reactions involving a wide range of substrates. Conventionally, lipases have been viewed as the biocatalysts for the hydrolysis of TAG (fats and oils) to free fatty acids, monoacylglycerols (MAG), diacylglycerols (DAG), and glycerol. The main advantages of lipase catalysis are selectivity, stereo-specificity, and mild reaction conditions. Despite these advantages and the fact that enzymatic splitting of fats for fatty acid production was described as early as in 1902, the lipase-catalyzed process has not replaced the commercial physicochemical process for the continuous splitting of TAG utilizing super-heated steam. The limited exploitation of lipase technology may be attributed to high enzyme cost, large reaction volume, requirement for emulsification of substrate, and risk of microbial contamination. Many of these limitations originate from the fact that lipases are employed mainly in water-rich reaction media where the solubility of the substrate TAG is very small. To circumvent this problem and to realize the full potential of lipase, researchers have explored newer approaches by manipulating the conditions under which the lipases act. Many of these novel approaches for lipase catalysis have been the outcome of the discovery that enzymes can be active in water-poor, non-polar media (Hanhan, 1952; Misiorowski and Wells, 1974; Zaks and Klibanov, 1984). Also, the finding that lipases can act in organic solvents has led lo an expansion of their applicability in a wide variety of chemical reactions. Lipase catalysis in some of the well established reaction media has previously been reviewed (Brockerhoff and Jensen, 1974; Brockman, 1984; Lilly et al., 1987; Hailing, 1990; Inada el al, 1990; Malcata et al., 1990). The present review is intended to present a compilation and comparison of novel reaction systems used for lipase catalysis. This review describes briefly the general characteristics of lipase reactions, applications of lipase in various fields, and conventional lipase technology. The lipase-mediated biochemical reactions, particularly the hydrolysis of TAG in novel reaction media is discussed in greater detail.
Organic carbonates are important classes of organic compounds and chemical intermediates, which are renowned for their great versatility, low toxicity, and high biodegradability. Therefore, they are widely acknowledged as green reagents and benign alternatives to conventional organic solvents. Today, most of the industrial technologies are compelled to implement the green processes to address both sustainability and eco-friendliness. In this context, the deployment of biocatalysis leading to high chemo-, regio-, and stereo-selectivities of the target products turns out to be an attractive solution. In this chapter, we will focus on a multiple of attempts to integrate the remarkable features of carbonates as green solvents and reagents in the biocatalyst-mediated reactions.
Abstract :
Along the last two decades, the biotechnology has become a challenging field of science, in
which the enzyme catalyzed racemic reactions play an important role in the production of
enantiomerically pure industrial compounds. Thus, “chiral switching” became an important
concept. The enzyme kinetics significantly takes good part in understanding the behavior of
the reaction, as well as determining the rate of reaction, by which it is later possible to design
a new bioreactor, and/or to optimize the reaction conditions.
In this work, first the enantioselective ratio (E-value) has been analyzed, while the general
aim is to increase the enantioselectivity of racemic reactions. The E value for the two
component (bi-bi) reactions was newly suggested according to the overall mechanism in the
bulk of the reaction medium.
In addition, a general mechanistic model has been proposed and solved by making a pseudosteady state assumption. A general rate expression for any racemic reaction has been derived
with a Maple software program. A specific reaction between isopropyliedeneglycerol and
vinylacetate, as acyl donor, was considered as a case study to test the proposed model. It is
obtained that the general model can easily be applied to this specific case, and a rate
expression was derived for this specific reaction. The rate constants were simulated with
random non-linear regression tool of Matlab software package.
This model has also been applied to single enantiomer, and it is found that the rate of reaction
becomes a simple Michaelis- Menten type, and the Michaelis- Menten constants for both
enantiomeres Vmax,DS( 0.677 mol/L.h ), Km,DS (0.285 mol/L ) Vmax,LS( 0.66 mol/L.h ), Km,LS
(0.98 mol/L ) were separately evaluated.
A new general E- value based on mechanistic model has been suggested for all cases, after
derivation of the general rate expression. This new equation was verified for the irreversible
case and it is found that it simply turns to be the E-value suggested in the literature for the
irreversible conditions.
As a result, the general model proposed in this study can be used for any type of racemic
enzymatic reactions. If the mechanism of a reaction can be defined depending on its
molecular interactions, then it is possible to determine the type of reactions between
intermediates. Accordingly, the reversible rate constants are equated to zero for the
irreversible reactions, and the model becomes case specific one, whose coefficients can be
calculated by computer programs like Matlab and Maple. The enantiomeric ratio based on
mechanistic model can be used in studies to calculate the enantioselectivity of the enyzmes.
vi
Keywords: Racemic reaction, transesterification, enantioselectivity, lipase,
kinetic modeling, organic solvent
Die Schlüsselwörter : racemischer Reaktion, Transesterifikation, Enantioselektivität,
lipase, kinetische Modellierung, Organische Lösung
Water is the most common solvent for biochemical reactions both in vivo and in vitro in enzymological experiments. Unfortunately, synthetic reactions that can be carried out by reversing the hydrolytic action of certain enzymes as well as other biotransformations (e.g., oxidation) are difficult or even impossible to operate in water. Oxidation reactions are limited by the poor solubility of oxygen in water, and syntheses are impeded by high water activity. In order to shift the thermodynamic equilibrium in favor of the synthesis, it is necessary to use nonaqueous solvents. However, the use of solvents can be problematic because of toxicity, flammability, and increasing environmental concerns. As a result, supercritical fluids (SCFs) have attracted much attention in recent years as an alternative to organic solvents for carrying out enzymatic reactions. Up to now, SCFs have only been used in large-scale industrial processing to extract plant materials (e.g., coffee, hops). Nevertheless, interest in the use of SCF as a solvent for biocatalysis is growing rapidly (see reviews in refs. 1-9).
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.
The Chapter outlines the main factors influencing enzyme activity and stability and the process parameters impact on reaction rates and productivity, on application of various types of reactors, and on limitations of enzymes applications as biocatalyst in supercritical fluids. Future trends of development are presented as well.
White biotechnology is the use of enzymes and microorganisms in industrial production through applied biocatalysis. This allows for milder reaction conditions (pH and temperature) and the use of more environmentally-compatible catalysts and solvents. This, in turn, leads to processes which are shorter, generate less waste, making them both environmentally and economically more attractive than conventional routes.
This book describes the use of white biotechnology within the sustainable chemistry concept, covering waste minimization; the use of alternative solvents (supercritical fluids, pressurized gases, ionic liquids and micellar systems) and energies (microwaves and ultrasound); sustainable approaches for the production of fine and bulk chemicals (aromas, polymers, pharmaceuticals and enzymes); the use of renewable resources and agro-industrial residues; and biocatalysts recycling.
Covering industrial processes and new technologies, this book combines expertise from academia and industry. It is a valuable resource for researchers and industrialists working in biotechnology, green chemistry and sustainability.
Hydrolysis of amylopectin by a debranching enzyme, isoamylase, was investigated under pressurized carbon dioxide in a reactor of one liter for various agitation rates and pH values. The experimental results indicated that agitation significantly inactivated the enzyme with pressurized carbon dioxide. The pH shift due to higher solubility of carbon dioxide in the system also decreased the rate. With proper tuning of agitation rate and pH, the enzyme hydrolysis reaction under supercritical carbon dioxide demonstrated better result than that under atmospheric conditions at higher substrate concentration.
Four classes of CO2-based solvents are described, with their distinctive properties and applications. Supercritical CO2, meaning CO2 above its critical temperature and pressure, requires high pressure but is nontoxic, has excellent mass transfer rates and is easy to remove from products. Liquid CO2 has many of the same benefits but at a lower temperature and pressure, although it is a particularly weak solvent. CO2-expanded liquids are organic liquids into which a large amount of CO2 has been dissolved under pressure; the CO2 content modifies and tunes many of the liquid properties. Switchable solvents can reversibly change their properties whenever needed, so that they can, for example, dissolve some material and then later release it upon application of a trigger; CO2 at atmospheric pressure is the trigger of choice.
Enzyme efficiency was investigated in the lipase-catalyzed hydrolysis of canola oil in supercritical carbon dioxide (SCCO2). Immobilized lipase from Mucor miehie (Lipozyme IM) was used as the catalyst and the results showed that enzyme efficiency dropped at high pressures indicating a possible change in enzyme microstructure. Therefore, scanning electron microscopy (SEM) was used as a supplementary tool to investigate microstructural stability of the enzyme under supercritical conditions. SEM images of the treated enzymes did not demonstrate any apparent structural changes with a change in pressure (24.0 and 38.0 MPa), enzyme load of the reactor (1.0 and 5.0 g), CO2 flow rate (0.5 and 3.9 L/min) and the oil content (extracted from 3.0 and 15.0 g canola flakes) of SCCO2. However, a change at the molecular level is a possibility, which requires further investigation.
BACKGROUND
In this work, CO2-based micelles with TMN series surfactants were used as reactive medium. Hydrolysis of p-nitrophenyl butyrate catalyzed by porcine pancreas was used as a model reaction. The reactive behavior was used to optimize the reaction temperature, pH, pressure, enzyme concentration and the added water.RESULTS AND DISCUSSIONGenerally, the reaction parameters depended on the types of substrates and enzymes. For the hydrolysis of p-nitrophenyl butyrate in CO2-based micelles, the reactive yield reached a high level at temperature 303 K, system pressure 15 MPa, pH 6.8, water to surfactant ratio W0 15, and enzyme concentration 1 g mL-1. In addition, the system reactivity was highest when TMN-6 surfactant was used.CONCLUSIONS
The activity of lipase-catalyzed hydrolysis in CO2-based micelle was increased significantly compared with that obtained in cyclohexane oil micelle. The combination of enzyme with supercritical CO2 represented a promising ‘green’ reaction system for bioconversions. © 2013 Society of Chemical Industry
New enzymatic reactions in supercritical fluid carbon dioxide catalyzed by lipases (PPL, Lipase MY, Candida cylindracea Lipase), and Proteases (subtilisin Carlsberg, subtilisin 8397, immobilized papain) with high efficiency and yields in a simple high pressure reactor using readily available dry‐ice have been developed.
The subtilisin Carlsberg catalysed transesterification of N-acetyl phenylalanine methyl ester (1), N-acetyl phenylalanine ethyl ester (2), N-trifluoroacetyl phenylalanine methyl ester (3) and N-trifluoroacetyl phenylalanine ethyl ester (4) was studied in supercritical carbon dioxide. The water content of the reaction affects the reactivity of the system; for the transesterification of the methyl esters with ethanol the optimum concentration of water was determined to be about 0.74 M, while for the transesterification of the ethyl esters with methanol the optimum concentration of water was about 1.3 M. The conversion is also dependent upon the concentration of alcohol; for ethanol, 2% v/v gives the maximum conversion, whilst for methanol, only 0.8-1.2% v/v is required. This is probably due to a difference in the solubility of the substrates in the two alcohol/supercritical carbon dioxide mixtures. The reaction is highly stereoselective, in all cases no evidence for reaction of the D-isomer could be detected by chiral gas chromatography.
The developments on applications of supercritical fluids as alternative solvents for biocatalytic processes that have taken place over the past two decades have been reviewed. An overview of process parameters influencing enzyme activity and stability, the influence of process parameters on reaction rates and productivity are presented. Applications of various types of reactors for enzymatic reaction in dense fluids, limitations of using enzymes as biocatalyst in supercritical fluids as well as future trends are presented. Main advantages of using dense gases as solvents for biocatalyzed reactions are the tunability of solvent properties and simple down stream processing features that can be readily combined with other unit operations. Although many enzymes are stable in supercritical fluids (SCFs) one should pay considerable attention to finding the correct reaction conditions for each substrate/enzyme/SCF system. One of the persistent problems is the instability and deactivation of enzymes under pressure and temperature. At present the most stable enzymes are hydrolases (lipases and esterases) for which pressure effect is lower than temperature deactivation.
The objective of the Patents and Literature Section ofApplied Biochemistry and Biotechnology is to summarize and cite recent developments in industrial and academic research as portrayed within the scope of recent patents and literature and to highlight emerging biotechnological research areas. To add to the value of this section of the journal, mini-reviews on a topic in biotechnology will be included. Following each mini-review, a patent and literature search will be presented, much as before. The reviews will be authored by experts in the topic, who will also prepare the patent and literature searches. The manuscripts will be peer-reviewed. Reviews will be solicited by the Patents and Literature editor, but unsolicited submissions are also encouraged. However, please contact the Patents and Literature Editor regarding a topic of interest before commencing with the preparation of such a review.
For information and/or suggestions for future topics, contact Jonathan S. Dordick, Editor, Patents and Literature, Department of Chemical and Biochemical Engineering, University of Iowa, 129 CB, Iowa City, Iowa 52242.
The application of supercritical fluids (SCFs) as reaction media for enzymatic synthesis has several advantages, such as the higher initial reaction rates, higher conversion, possible separation of products from unreacted substrates, and over solvent-free or solvent systems. An additional benefit of using SCFS as reaction media is that they give simple and ecologically safe (no heat and solvent pollution) recovery of products. However, for some specific reactions, solvent-free systems are preferred because of their higher yields. The main area of development should be related to the hydrolysis of glycerides, transesterification, esterification, and inter-esterification reactions. As lipases have high and stable activity in supercritical carbon dioxide (SC CO2)—even at the high temperature—an intensive development is expected. The advantages of using SC CO2 as a medium for enzyme-catalyzed reactions have been well documented. Frequently, the temperature ranges used for employing supercritical carbon dioxide in processing are compatible with the use of enzymes as catalysts. An additional benefit of using supercritical fluids along with enzymatic catalysis is that it provides a medium for the recovery of products or reactants.
The processing parameters in enzymatic reactions using CO2-expanded (CX) lipids have strong effects on the physical properties of liquid phase, degree of interesterification, and physicochemical properties of the final reaction products. CX-canola oil and fully hydrogenated canola oil (FHCO) were interesterified using Lipozyme TL IM in a high pressure stirred batch reactor. The effects of immobilised enzyme load, pressure, substrate ratio and reaction time on the formation of mixed triacylglycerols (TG) from trisaturated and triunsaturated TG were investigated. The optimal immobilised enzyme load, pressure, substrate ratio and time for the degree of interesterification to reach the highest equilibrium state were 6% (w/v) of initial substrates, 10MPa, blend with 30% (w/w) of FHCO and 2h, respectively. The physicochemical properties of the initial blend and interesterified products with different FHCO ratios obtained at optimal reaction conditions were determined in terms of TG composition, thermal behaviour and solid fat content (SFC). The amounts of saturated and triunsaturated TG decreased while the amounts of mixed TG increased as a result of interesterification. Thus, the interesterified product had a lower melting point, and broader melting and plasticity ranges compared to the initial blends. These findings are important for better understanding of CX-lipid reactions and for optimal formulation of base-stocks of margarine and confectionary fats to meet industry demands.
This chapter discusses the enzymatic catalysis in supercritical carbon dioxide (scCO2). scCO2 offers some advantages over organic solvents. It is not flammable, non toxic, and relatively cheap and offers the possibility of an integrated separation process. To take advantage of the stereoselectivity of enzymatic catalysis, this chapter describes the use of transesterification of menthol as model system. Esterase EP10 from Pseudomonas marginata proved to be a suitable catalyst for the production of enantiomerically pure menthyl acetate. The water content of the reaction medium is an important factor governing the reaction rate of the transesterification. Various groups have investigated the role of water content on reaction velocity. The chapter describes the use of aluminum oxide humidity sensor to measure inline, the water vapor pressure. The partitioning of water between scCO2 and the enzyme preparation is examined and the influence of water activity on the initial reaction velocity is determined in the chapter.
The thermal stability and stability against pressurization/depressurization steps of lipase from Pseudomonas sp. and lipase from Candida cylindracea have been compared with data of other enzymes previously published. Hydrolases (lipases and esterases, crude preparations) with disulfide bridges have a lower degree of inactivation after several pressurization/depressurization steps but no better thermal stability (75°C, 150 bar, 24 h) compared to an enzyme without cystines. A higher loss in enzyme activity was observed after 30 depressurization steps from the liquid phase (56.3% loss of the initial activity) than from the supercritical phase (36.1% loss). Twenty phase transition cycles (liquid–supercritical and supercritical–liquid) do not cause more enzyme inactivation (86.8% residual activity) than simple incubation at 20°C (95.3%) or 65°C (82.1%). Comparing a crude form of lipasefrom Aspergillus niger and a preparation with higher activity, it turns out that the stability of the crude enzyme (92% of initial activity) against 30 pressurization/depressurization steps is similar to that of the purer preparation (102%). Also, thermal stabilities do not differ greatly (108% vs. 97%). Fluorescence spectra indicate no conformational change oflipase from Aspergillus niger after SC-CO2 treatment.
This article provides a bibliographic listing of journal papers published between 1980–1993 concerned with supercritical extraction. This paper provides coverage of a topic of relatively recent interest (since 1980) but which is growing rapidly in terms of applications and the number of publications each year. Supercritical extraction (often using carbon dioxide) has been used for the extraction of caffeine from coffee beans, and removal of aromatics and oils from plant materials. Recent interest has centered on environmental problems and it is currently being investigated as a possible technique for the separation and recycling of polymer materials. Other applications are emerging in the biotechnology area. This bibliography is timely in that it provides a quick and easy reference source, and a more detailed coverage than has been provided in other bibliographic papers (1–3). The following topics are included in this paper:
A continuous flow apparatus is set up to study the enzymatic interesterification of palm oil by stearic acid in supercritical carbon dioxide. Domestic edible palm oil is mixed with stearic acid and loaded to a saturation tank in which the oil is extracted by supercritical carbon dioxide. The current of the extraction solution bubbled from the saturation tank is delivered to an enzyme-packed column. In this study, five triglycerides (POP, POS, POO, OOO, and SOO) and two free fatty acids (stearic acid and palmic acid) are selected as indicators to monitor the interesterification. The results show that the Mucor miehei dominantly catalyzes the interesterifications of POP+S«POS and POO+S«SOO when the stearic acid content in the extraction solution is abundant. A very limited amount of SOS is also irregularly found in the product samples. POS and SOO are rarely produced when the loaded stearic acid is completely elutriated, and the weight fraction of POO is significantly increased by the depletion of POP. It is presumed that the palmitoyl group in the 1,3 position is substituted much more readily, and that the stearic acid is a reactive acyl donor for the interesterification in supercritical carbon dioxide when catalyzed by Mucor miehei. That large amounts of palmic acid are found in the transesterified oil confirms this presumption.
The enzymatic estenfication of myristic acid with ethanol catalyzed by an immobilized lipase from Mucor Miehei has been performed under supercritical conditions.After previous experiments in supercritical carbon dioxide showing a high stability of this enzyme1 and leading to a kinetic description of the reaction in a stirred reactor,2 the reaction was realized in a continuous mode through a fixed-bed reactor.The effects of water concentration and fluid flowrate on the reaction rate are investigated. An increase of carbon dioxide moisture content from 0 to 0.25% (mass) causes an increase in the conversion, whereas, beyond this value, the lipase activity is irreversibly altered. Production rate surprisingly increases with fluid flowrate when reactants flowrates are maintained constant, but decreases when reactant concentrations are kept constant, confirming a reaction mechanism including inhibition by ethanol.
Alkaline protease “alcalase” has been found to be very stable and active in supercritical carbon dioxide. Procedures for resolution of N-protected amino acid derivatives in supercritical carbon dioxide catalyzed by alcalase with high yields and optical purity have been developed.
The acylation of glucose with lauric acid in a reaction catalysed by two Candida lipases and a Mucor miehei lipase in supercritical carbon dioxide (SCCO2) was investigated. A linear dependence of the reaction rate on enzyme concentration was observed. Studies on the effect of temperature on enzyme activity showed that Candida antarctica lipase remains stable at temperatures as high as 70°C. Non-immobilised Candida rugosa lipase was found to have a temperature optimum at 60°C. The acylation reaction rate depended on the initial water activity of both substrates and enzyme; the optimum was 0·75 for Candida antarctica lipase, 0·53 for Candida rugosa lipase, and between 0·3 and 0·5 for Mucor miehei lipase. Candida rugosa lipase was most active at a molar ratio of sugar: acyl donor of 1: 3, while the optimum ratio was found to increase to 1: 6 when the reaction was catalysed by Candida antarctica and Mucor miehei lipases. © 1998 SCI
This chapter discusses the enzymatic reactions in supercritical carbon dioxide. The intrinsic catalytic properties of enzymes are modified either during immobilization or after they are immobilized. In heterogeneous catalysis, carried out by immobilized enzymes, the rate of reaction is determined not simply by pH, temperature, and substrate solution but by the rates of proton, heat, and substrate transport through the support matrix to the immobilized enzyme. The chapter discusses the internal mass transfer limitation both in hexane and in supercritical CO2 (scCO2) with different enzymatic support sizes. The theory of generalized Thiele modulus can be applied to an enzymatic reaction both in n-hexane and scCO2. The Thiele modulus values indicate a limitation because of the internal mass transfer rate. Thus, in the hexane case, a diffusional control is observed, while in scCO2, an intermediate rate between the reactional and diffusional rates is apparent.
N-vanillylnonanamide (VAN) was successfully synthesized from vanillylamine hydrochloride by enzymatic catalysis in supercritical carbon dioxide (SC–CO2). Five commercial lipases, Novozyme 435, Lipozyme IM, Amano PS, Amano G and Sigma Candida cylindracea type VII, as biocatalysts for VAN synthesis were compared. Lipozyme IM exhibited best yields of tested lipases. Various parameters such as time, temperature, pressure and vanillylamine hydrochloride/nonanoic anhydride ratio that influenced the reaction were investigated. Nonanoic anhydride showed the best acyl donor of the employed substrates. An amidation yield of 40% was obtained when nonanoic anhydride and Lipozyme IM were used at 170bar and 50°C for 23h in SC–CO2. Besides, addition of 2mM divalent salts (CuCl2 and ZnCl2) significantly increased 11–23% yield of the VAN. The enzyme operational stability suggested that Lipozyme IM maintained over 50°C of the initial activity for the synthesis of VAN after reuse for 69h. Furthermore, in vitro, VAN behaved as a potential antibacterial against Escherichia coli.
Activity of free Candida rugosa lipase (CRL) in hydrolysis reaction of tuna oil was reduced significantly under high pressure carbon dioxide (CO 2). A lot of factors, such as water content, pressure, temperature, pH, phase behavior and the kind of atmospheric gas affect the activity of CRL in the hydrolysis reaction under high pressure CO 2 and the effect of them was investigated. The hydrolysis activity was affected mainly by the water content, pressure and temperature. Besides, pH was not a key factor for the hydrolysis activity of CRL under high pressure CO 2. The dilution effect by a great amount of CO 2 dissolved in the oil phase was suggested to be the most possible cause to the reduction of hydrolysis reaction activity of CRL under high pressure CO 2.
Dense gases were used as a biochemical reaction medium. Esterification of oleic acid with oleyl alcohol, catalyzed by lipase from Rhizomucor miehei (LipozymeIM) was used as a model system. Due to the limitation of the process that may arise from the non-polarity of carbon dioxide, which preferentially dissolves hydrophobic compounds, studies were also performed with other gases (n-butane, n-propane, n-propane/n-butane mixture).The study of the pressure stability of the immobilized lipase showed that the lipase is quite stable; it does not lose its activity when it is exposed to various dense gases at high pressure for a longer time. Esterification rates at high pressure were determined for all systems, and it was found that they were higher than at atmospheric pressure. The studies of thermodynamic properties and mass transfer were performed in a batch stirred tank reactor in dense carbon dioxide for the esterification of oleic acid with oleyl alcohol (catalyzed by lipase). The highest rate and maximum conversion were determined.It was found that in a continuous fixed bed reactor at 150bar, 40°C and water activity 0.46% w/w, the activity of the enzyme preparation was practically unchanged when CO2 was used as a solvent. The addition of small amounts of water increases the conversion rate. A higher conversion was also observed at a longer residence time. When n-butane was used as a reaction medium a decrease of conversion was observed.
The sections in this article are
7.1 Enzymes in Non-aqueous Environments
7.2 Supercritical Fluids for Enzyme Catalysis
7.3 Enzymatic Reactions in Supercritical Fluids
7.4 Reaction Parameters in Supercritical Biocatalysis
7.5 Stabilized Enzymes for Supercritical Biocatalysis
7.6 Enzymatic Catalysis in IL–scCO2 Biphasic Systems
7.7 Future Trends
7.8 Acknowledgments
This chapter reviews the field of reactions in supercritical fluids (SCFs) and gives several examples of experimental studies on lipase-catalyzed reactions in SCFs. Supercritical fluids can be advantageously used as reaction media. It may be possible to carry out enzymatic reactions reducing interphase mass transfer limitations, and labile reaction products could be more readily isolated from the reaction mixture by adjusting the pressure and/or the temperature to induce a phase split, thus avoiding unwanted side reactions. Moreover, reaction rates may be advantageously enhanced by running the reaction at conditions close to the critical point of the pure SCF. A change in temperature or pressure can influence the solvation state of the substrate, product, and even the enzyme, and improve results in terms of reaction velocities and selectivity. Integration of reaction and separation bioprocesses will further demonstrate the superior characteristics of SCFs and will lead to use of these fluids in many new application areas.
A kinetic study of ethyl myristate formation by esterification of myristic acid with ethanol catalysed by a lipase from Mucor miehei immobilized on Duolite A 568, has been carried out. The reaction was done in a two liquid phase system in which the non-aqueous solvent was supercritical carbon dioxide (P= 12.5 MPa, T = 323 K) or n-hexane (P = 0.1 MPa, T = 323K). In heteregenous catalysis, it is important to study the internal mass transfer limitation. For this, the support was sieved into different size series (from 300 to 600 mm) and the reaction was carried out for each size series. The samples were analyzed by gas chromatography and the esterification was shown to follow Ping Pong Bi Bi kinetics with competitive substrate inhibition. The Thiele modulus values show that there is, in the hexane case, a diffusional control while in SCCO2 media, we obtained an intermediate rate between reactional and diffusional rates.
Abstract Supercritical fluids are materials above their critical point that represent a unique class of nonaqueous media for biocatalysis and bioseparation. The inherent gas-like low viscosities and high diffusi vities of supercritical fluids increase the rates of mass transfer of substrates to enzyme. Conversely, the liquid-like densities of supercritical fluids result in higher solubilizing power than those observed for gases. Unlike the behavior of gases and liquids, the physical properties of a supercritical fluid can be adjusted over a wide range by a relatively small change in pressure or temperature. In a supercritical fluid, the careful regulation of the density enables reactant and product solubility to be controlled, thus simplifying downstream separations. The extraction power of supercritical carbon dioxide has been used extensively in both the chemical and food industries. The use of supercritical fluids as a dispersent for biocatalysis was first described in 1985, and there is now a growing trend in using supercritical fluids as reaction media for enzymatic catalysis. The advantages of using enzymes in supercritical fluids include the following: 1. Synthesis reactions in which water is a product can be driven to completion. 2. The solubilities of hydrophobic materials are increased relative to those in water. 3. The thermostability of biomolecules in supercritical fluids is greater than in water. 4. The solvent can be readily recycled. 5. Biochemical reactions and separations can be integrated into a single step. Among potentially interesting solvents for enzymatic catalysis, carbon dioxide is the most widely used supercritical fluid. However, there is a growing interest in using other supercritical fluids (e.g., ethylene, fluoroform, ethane, sulfur hexafluoride, and near-critical propane). In this review, we focus on describing enzymatic catalysis in supercritical fluids performed to date, and we address the fundamental issues associated with supercritical fluid-based biocatalysis.
We report on the effects of O2 on the luminescence quenching of ruthenium(II) tris(2,2‘-bipyridyl) cation, [Ru(bpy)32+], sequestered within the water pool region of perfluoropolyether (PFPE)-based reverse micelles formed in supercritical carbon dioxide (scCO2). A Stern−Volmer quenching model cannot describe the O2-dependent [Ru(bpy)32+] quenching profiles in the PFPE micelles. A “two-site” model is needed to describe the observed quenching. The mean quenching efficiency is 50-fold higher when the molar ratio of water to PFPE (ωo) is ≤10 as compared to ωo = 20. At ωo values of 10 or less, 95+% of the [Ru(bpy)32+] molecules within the water pool are located in close proximity to the carboxylate headgroups within the micelle water pool and they are quenched very effectively by the O2 dissolved within the scCO2. At the highest ωo values studied, 60−65% of the [Ru(bpy)32+] within the water pool remains near the carboxylate headgroups; a substantial fraction (35−40%) of the [Ru(bpy)32+] is distributed more toward the center of water pool away from anionic surfactant headgroups. Those [Ru(bpy)32+] luminophores that are away from the headgroups (more toward the water pool center) are not quenched as effectively by O2 because the O2 solubility in water is significantly lower compared to scCO2.
Lipase-catalyzed syntheses of oleic acid esters with various primary alcohols have been performed in a batch stirred tank reactor in an almost nonaqueous medium without organic solvents. For all syntheses 50 °C was found to be the optimal temperature. Initial reaction rates were influenced by the alcohol chain length. The study of the pressure stability of the immobilized lipase from Rhizomucor miehei (lipozyme IM) showed that the lipase preparation is quite stable; it does not lose its activity when it is exposed to carbon dioxide at 300 bar for 24 h. Esterification rates at high pressure were determined, and it was found that they were higher than at atmospheric pressure. The highest rate and maximal conversion were near the critical point of carbon dioxide. Keywords: Esterification; batch reactor; lipase; Mucor miehei; oleic acid esters; high pressure; carbon dioxide; enzyme stability
Cyclohexane oxidation has been studied in supercritical carbon dioxide medium for homogenizing the initial reaction mixture to produce cyclohexanone and cyclohexanol as the chief reaction products. The kinetic experiments have been performed at three temperatures 410, 423, and 433 K and two pressures 170 and 205 bar. The results have been interpreted in the light of transition state theory and cage effects. Conversions obtained are low compared to the liquid phase oxidation because of dilute concentrations of the reactants. Cyclohexanone is more selectively formed and favored by both pressure and temperature. A 20% increase in pressure results in (1) reduction of the induction period by 50%, (2) a change in activation energy from 13.0 kcal/mol at 170 bar to 22.6 kcal/mol at 205 bar, (3) an increase in the preexponential factor by 5 orders of magnitude, and (4) an increase in the first-order rate constant at 433 K by about 70%. The variation in the observed activation volume from 36 cm[sup 3]/mol at 410 K and 170 bar to [minus]775 cm[sup 3]/mol at 433 K and 205 bar suggests that the reaction in supercritical CO[sub 2] medium can be greatly manipulated.
IntroductionEnzymesEnzyme ReactorsExperimental ResultsDownstream Processing and CostsSummary and Outlook
Availability and Structure of LipasesWater, Organic Solvents, and Other Reaction MediaEnantioselective ReactionsChemo- and Regioselective ReactionsCommercial Applications and Future DirectionsReferences
This work investigated the immobilised lipase kinetics of esterification of oleic acid and ethanol. The reaction was conducted under supercritical conditions (13 × 106 Pa and 40 °C) using carbon dioxide as solvent in a continuous packed bed (plug flow) reactor. Biocatalyst LypozymeTM IM60, which is lipase from Rhizomucor miehei (EC.3.1.1.3), immobilised on Duolite (anionic exchange resin) was used as biocatalyst. Kinetically, with regard to oleic acid, the reaction was successfully modelled by the Michaelis–Menten mechanism. The reaction rate constants Km and Vmax were evaluated. Furthermore, it was found to undergo competitive inhibition by ethanol, and the inhibition constant Ki was evaluated.© 2000 Society of Chemical Industry
Summary The stability of the monomeric enzymes a-chymotrypsin and trypsin, and the oligomeric enzyme penicillin amidase in supercritical CO2 has been studied. They were found to be partly denatured during the depressurization step. The degree of denaturation was larger in humid CO2 than in dry CO2. Enzymes with S-S bridges (a-chymotrypsin; trypsin) were denatured to a lesser degree than the enzyme without cysteine (penicillin amidase). These results and electrophoretic and spectroscopic analysis indicated that the denaturation was caused by partial unfolding during the depressurization step.
The author has examined about a dozen different enzymes for their ability to act as catalysts in organic media and has yet to find one unable to do so. Because those enzymes are unrelated in terms of their structure, size, origin, and function, and because they consist of the same 20 common amino acids as all other enzymes, there is no reason to believe that they are unique. Therefore, it seems safe to say that most, if not all, enzymes can work in organic solvents. This conclusion should have far-reaching implications for the practical use of enzymes. Applications that have been rules out because of enzymes' shortcomings as industrial catalysts should be reevaluated in light of the feasibility of their use in organic solvents instead of water.
In this review article, the following topics are discussed: the background; phase equilibrium considerations (binary mixtures and ternary mixtures); theory and data correlation; a tabulation of experimental studies; transport coefficients (diffusion coefficients, viscosity, Schmidt numbers, and mass transfer coefficients); and applications such as the separation of chemicals from water streams, processes for coffee and tea decaffeination, oil extraction from seeds and foods, the processing of low-vapor pressure oils, and the extraction of solvents, monomers, and fractionation of polymers, as well as the current status of applicational development.
Microreaction technology is a new continuous processing concept with different types of reactors based on microengineered structures. These extend the performance of conventional reactors especially in terms of enhanced mass and heat transfer, for example, to be used for fast exothermic reactions and safe operation under extreme processing conditions and with hazardous reagents. The hydrodynamics of gas/liquid microreactors is often characterized by uniform flow patterns such as the Taylor flow. Some gas/liquid microreactors are just miniaturized analogues of their macroscale counterparts, for example, the falling-film microreactor, whereas others offer entirely new multiphase contacting concepts, for example, the Taylor-flow or mesh microreactors. In the following, some major contacting principles will be given, which will be first accompanied by realized reactor examples. Then, the application of the microreactors for gas/liquid and gas/liquid/solid reactions in the field of organic chemistry will be presented and will cover the major part of this chapter.
The use of a supercritical fluid as a medium for enzymatic reaction was investigated with respect to its application and reaction mechanism. Lipase could catalyze the reaction of hydrolysis and interes-terification in supercritical carbon dioxide at 35°C and 137 bar. There was no difference in the time course of interesterification between the untreated lipase and the lipase exposed to supercritical carbon dioxide indicating that the enzyme was not severely damaged.
Dense (supercritical) gas chromatography and the related rapidly growing field of dense gas extraction are reviewed and an extensive compilation detailing the dense gas systems (solvent gases, solutes, temperatures, pressures) that have been studied is presented. Furthermore, dense gas chromatography is compared to both gas and high performance liquid chromatographies with emphasis on mass spectrometric detection in all three. “It is concluded that a dense gas chromatograph/mass spectrometer, a new instrument, is both a complement to existing techniques and a timely development for use with dense (supercritical) gas systems.”
The objective of this paper is to present a review of the field of reactions in supercritical fluids (SCF's). The high pressure phase behavior of mixtures in their critical region has a direct bearing on the understanding and interpretation of kinetic rate data, and, therefore, a discussion of high pressure phase behavior is presented. Mention is made of transition-state analysis as applied to SCF reaction studies. Further, the unusual partial molar volume behavior of a heavy solute solubilized in an SCF solvent is described and related to the enhancement of the reaction rate near the critical point of the SCF. Several examples of experimental studies of reactions in SCF media are presented, and the advantages of an SCF reaction scheme as compared to a conventional reaction scheme are described. Finally, some observations are made based on our own experience as to the potential of this emerging technology.
The theory of diffusion and simultaneous chemical combination of two gases dissolving in a liquid and reacting near the interface is discussed mathematically. The nonlinear partial differential equations representing mass balances and corresponding to the penetration theory are solved numerically. The chemical reaction rate expression is second order, corresponding to a bimolecular mechanism. The results show that chemical reaction between the two dissolved substances increases the rate of absorption of each by an amount that depends on the ratio of solubilities, the reaction rate constant, and the diffusion coefficients. Mathematical solutions are also presented for the simultaneous absorption of two gases in a liquid which contains a third component capable of reacting chemically with both of the dissolved gases.
In this paper the University of California, Berkeley, Sulfur Recovery Process (UCBSRP) is compared to conventional technology for the case of the removal of H2S from the recycle gas of a high-pressure petroleum residuum hydrotreater. The conventional technology selected for this comparison consists of an absorber/stripper operation using diethanol amine as the absorbent, a Claus sulfur plant, and a SCOTT tail-gas treating unit. Flowsheets, stream flows and conditions, and the total purchased cost of the major items of equipment are presented for both processes. From this comparison it is estimated that the direct fixed capital (DFC) for the UCBSRP would be about 61% of that for the conventional technology. The utility costs for this application of the UCBSRP are estimated to be less than the credit for the high-pressure steam produced whereas the utility costs for the conventional process are substantially more.
The development of biocatalytic reactors for the conversion of high concentrations of water-insoluble reactants is at the same stage that systems for water-soluble reactants had reached almost a decade ago. There are still many problems and much research to be done before we fully understand such systems. Nevertheless, we can confidently expect to see new commercial processes with multiliquid biocatalytic reactors during the next decade.
The enzyme alkaline phosphatase, EC 3. 1. 3. 1, was found to be active in a supercritical carbo dioxide solvent system. A batch reaction of disodium p-nitrophenyl phosphate with a 0.1 vol. % water solution in supercritical CO2 at 100 atm and 35C produced p-nitrophenol when catalyzed by alkaline phosphatase.
The enzyme polyphenol oxidase has been found to be catalytically active in supercritical carbon dioxide and fluoroform: it
readily oxidizesp-cresol andp-chlorophenol to their correspondingo-benzoquinones.
Extracellular microbial lipases can be used as catalysts for the interesterification of oils and fats. Use of specific lipases
gives products which are unobtainable by chemical interesterification methods. Some of these products have properties of value
to the oils and fats industry. The catalysts for enzymatic interesterification are prepared by coating inorganic support materials
with the lipases. For batch interesterification reactions, the catalyst particles are activated by addition of a small amount
of water and then stirred with a reactant mixture dissolved in petroleum ether. At the end of the reaction period, the catalyst
particles are removed by filtration, and the interesterified triglycerides isolated by conventional fat fractionation techniques.
The catalyst can be used in subsequent batch reactions. As an alternative to the batch reaction system, continuous enzymatic
interesterification processes can be operated by pumping water containg feedstock through a packed bed of activated catalyst.
Extraction with compressed fluids in the critical region is discussed in terms of the marked effect on solvent properties that can be brought about by small changes in pressure or temperature. The theoretical background and experimental data are described, including the classification of the phase behaviour of binary systems. A number of application studies are quoted, and comparison is made with liquid solvent extraction and distillation. Apart from such topics as the breaking of azeotropes, the main area of study is in performing separations on the basis of volatility where the general level of volatility is low. In the field of natural products these include the removal of undesirable substances such as caffeine and nicotine and the isolation of valuable constituents such as food essences and drugs. For fossil fuels, applications are described in enhanced oil recovery, fractionation of heavy petroleum liquids and extraction of liquids from coal.
The kinetics of interesterification reactions catalysed by Candida cylindracea lipase in cyclohexane have been investigated. The enzymatic rate constants Km and Vmax have been measured for the hydrolysis of trilaurin and the esterification of dilaurin and lauric acid, in the absence of mass transfer limitations. The enzymatic turnover number for esterification is three times higher than that for hydrolysis, suggesting that hydrolysis may be the rate-controlling step in interesterification. Equations are presented for Km and Vmax in terms of elementary-step rate constants.
Purified nonspecific lipase from rat pancreas appears to mediate the following reaction sequence: (1) HE + nB ⇌ HEBn; (2) HEBn + S ⇌ HEBnS; (3) HEBnS ⇌ EBnS' + ROH; (4) EBnS' + H2O → HEBn + R'COO− + H+, where E = enzyme, B = bile salt, and S = R'COOR. Evidence is presented for the occurrence, sequence and reversibility or irreversibility of the above four reactions. Also discussed are the activation energies for both a “good” and a “poor” substrate, the correlation between V and substituent function (σ) for a series of substituted benzoic acid esters, and the effects of steric hindrance. Studies of the incorporation of label from H218O further detail the hydrolytic mechanism, and the effects of the physical state in which substrate is presented to the enzyme (micelles, emulsion, crude bulk phase, etc.) are discussed.
Three grades of diatomaceous earth (Celite 560, Filtercel and Hyflo Supercel) and a controlled-pore silica have been examined for their suitability as support materials for lipase (triacyglycerol acylhydrolase, EC 3.1.1.3) catalysing the interesterification of fats. The controlled-pore silica gave a preparation with a low activity. Although all three Celites gave preparations with similar lipolytic activities, Hyflo Supercel gave the highest interesterification activity. The distribution of enzyme protein in Hyflo Supercel was examined by transmission electron microscopy.
The problem of gas absorption accompanied by an irreversible second-order reaction with a volatile reactant is considered. An approximate analytical solution for the reaction factor has been derived on the basis of the film theory and has been compared with the numerical solution. It is shown that the present approximate solution is in good agreement with the numerical solution.
Three different authors have reported on the use of four different enzymes in supercritical fluids. Lipase carries out transesterification reactions in the presence of supercritical carbon dioxide. Polyphenyl oxidase is active in supercritical COâ and fluoroform. It has been shown that alkaline phosphatase and cholesterol oxidase are active in supercritical COâ. More recently, an examination of the effect of aggregation of cholesterol on cholesterol oxidase activity in COâ using electron paramagnetic resonance (EPR) was done. They found that when cosolvents which promoted aggregation were added, the reaction rate increased in proportion to the amount of aggregation. To date, no data on the effect of pressure on reaction rate have been presented. The objective of this work is to determine whether pressure-induced changes in the physical properties of a supercritical fluid solvent affect the rate of an enzymatic reaction and if so, which properties are responsible for the change.
Subtilisin and alpha-chymotrypsin vigorously act as catalysts in a variety of dry organic solvents. Enzymatic transesterifications in organic solvents follow Michaelis-Menten kinetics, and the values of V/Km roughly correlate with solvent's hydrophobicity. The amount of water required by chymotrypsin and subtilisin for catalysis in organic solvents is much less than needed to form a monolayer on its surface. The vastly different catalytic activities of chymotrypsin in various organic solvents are partly due to stripping of the essential water from the enzyme by more hydrophilic solvents and partly due to the solvent directly affecting the enzymatic process. The rate enhancements afforded by chymotrypsin and subtilisin in the transesterification reaction in octane are of the order of 100 billion-fold; covalent modification of the active center of the enzymes by a site-specific reagent renders them catalytically inactive in organic solvents. Upon replacement of water with octane as the reaction medium, the specificity of chymotrypsin toward competitive inhibitors reverses. Both thermal and storage stabilities of chymotrypsin are greatly enhanced in nonaqueous solvents compared to water. The phenomenon of enzymatic catalysis in organic solvents appears to be due to the structural rigidity of proteins in organic solvents resulting in high kinetic barriers that prevent the native-like conformation from unfolding.
Fundamental studies of enzyme-solvent interactions can be conducted with supercritical fluids because small changes in pressure
or temperature may bring about great changes in the properties of a single solvent near its critical point. Cholesterol oxidase
is active in supercritical carbon dioxide and supercritical carbon dioxide-cosolvent mixtures. Variations in solvent power
caused by pressure changes or by the addition of dopants affected the rate of enzymatic oxidation of cholesterol by altering
the structure of cholesterol aggregates.
Vapor-Liquid Equilibria of Sulfur Dioxide in Polar Organic Solvents Numerical Simulation of Theories for Gas Absorption with Chemical Reaction
- R J Demyanovich
- S Lynn
Demyanovich, R. J.; Lynn, S. Vapor-Liquid Equilibria of Sulfur Dioxide in Polar Organic Solvents. Znd. Eng. Chem. Res. 1987, 26, 548. Glasscock, D. A,; Rochelle, G. T. Numerical Simulation of Theories for Gas Absorption with Chemical Reaction. AZChE J. 1989,35, 1271.
Received for review The interesterification of trilaurin (TL) and myr-istic acid (MA) to product 1,2(2,3)-dilauroyl-3(l)-myris-toyl-rac-glycerol (LLM), and 1,3-dimyristoyl-2-laurin (MLM) is believed to proceed via six steps
- R K Gupta
- E-Llm Q E + Llm
Gupta, R. K. Mass Transfer Characteristics of Plate Columns without Downcomers. Trans. Znst. Chem. Eng. 1967,45, T169. Stevens, C. A. Ph.D. Dissertation, University of California a t Berkeley, 1989. Received for review June 25, 1990 Revised manuscript received November 13, 1990 Accepted December 3, 1990 942 Ind. Eng. Chem. Res., Vol. 30, No. 5, 1991 1976). The interesterification of trilaurin (TL) and myr-istic acid (MA) to product 1,2(2,3)-dilauroyl-3(l)-myris-toyl-rac-glycerol (LLM), and 1,3-dimyristoyl-2-laurin (MLM) is believed to proceed via six steps: E + TL * E-TL E-LA + DL (1) E-LA + H20 -E-LA E + LA (2) E + MA w E-MA Q E-MA + H2O (3) E-MA + DL w E-LLM Q E + LLM Ind. Eng. Chem. Res.
Evaluation of Plate Efficiency for Ab-sorption with Chemical Reaction
- R Pohorecki
- W Moniuk
Pohorecki, R.; Moniuk, W. Evaluation of Plate Efficiency for Ab-sorption with Chemical Reaction. Znz. Chem. Procesowa 198313, 4, 353.
Enzymatic Synthesis of Nonyl-acetate and Isoamylacetate in Supercritical Carbon Dioxide and Organic Solvents
- R Stoop
Stoop, R. Enzymatic Synthesis of Nonyl-acetate and Isoamylacetate in Supercritical Carbon Dioxide and Organic Solvents. In Proceedings of the 2nd Netherlands Bio-technology Congress; Breteler, H., van Lelyveld, P. H., Luyben, K. Ch. A. M., Eds.; Netherlands Biotechnological Society: Zeist, The Netherlands, 198813.
Enzymatic Transesterification in Supercritical Carbon Dioxide
- R A Johnson
- N E Lloyd
- A M M Us Eijs
- J P J Dejong
- H J Doddema
- D R Lindeboom
- M A Visser
Johnson, R. A.; Lloyd, N. E. US. Patent 3788945, 1974. van Eijs, A. M. M.; deJong, J. P. J.; Doddema, H. J.; Lindeboom, D. R. Enzymatic Transesterification in Supercritical Carbon Dioxide. In Proceedings of the International Symposium on Supercritical Fluids; Perrut, M., Ed.; Societe Francaise de Chimie: Paris, 1988a. van Eijs, A. M. M.; deJong, J. P. J.; Oostrom, H. H. M.; Doddema, H. J.; Visser, M. A.;
Supercritical Fluid Extraction of Plant Materials Containing Chemotherapeutic Drugs
- V J Krukonis
- A R Branfman
- M G Broome
Krukonis, V. J.; Branfman, A. R.; Broome, M. G. Supercritical Fluid Extraction of Plant Materials Containing Chemotherapeutic Drugs. Presented at the AIChE Meeting, Boston, MA, Aug 1979.
Method for the Production of Caffeine-Free Coffee Extract
- W Roselius
- P Hubert
Roselius, W.; Vitzthum, 0.; Hubert, P. Method for the Production of Caffeine-Free Coffee Extract. U S. Patent 3,843,824, 1974. Subramaniam, B.; McHugh, M. A. Reactions in Supercritical Fluids-A Review. Ind. Eng. Chem. Process Des. Deu. 1986, 25, 1-12.
An Integrated Process for Simultaneous Desulfurization, Dehydration and Recovery of Hydrocarbon Li-quids from Natural Gas Streams Gas absorption with chemical reaction: the case involving a volatile liquid reactant
- S F Sciamanna
- S Lynn
- H2s Solubility
- Soz
- Coz
- Propane
- S F Sciamanna
- S Lynn
Sciamanna, S. F.; Lynn, S. Solubility of H2S, SOz, COz, Propane, and n-Butane in Polyglycol Ether. Znd. Eng. Chem. Res. 1988a, 27, 492. Sciamanna, S. F.; Lynn, S. An Integrated Process for Simultaneous Desulfurization, Dehydration and Recovery of Hydrocarbon Li-quids from Natural Gas Streams. Znd. Eng. Chem. Res. 1988b, 27,500. Shaikh, A. A.; Varma, A. Gas absorption with chemical reaction: the case involving a volatile liquid reactant. Chem. Eng. Sci. 1984, 39, 1639.
Kinetics of the Reaction of H a and SO2 in Organic Solvents Gas absorption with reaction in a solution con-taining a volatile dissolved reactant
- D W Neumann
- S Lynn
Neumann, D. W.; Lynn, S. Kinetics of the Reaction of H a and SO2 in Organic Solvents. Znd. Eng. Chem. Process Des. Deu. 1986,25, 248. Pangarkar, V. G. Gas absorption with reaction in a solution con-taining a volatile dissolved reactant. Chem. Eng. Sci. 1974, 29, 877.
Supercritical COz Extraction of Lipids from Lipid-Containing Materials
- J P Friedrich
Friedrich, J. P. Supercritical COz Extraction of Lipids from Lipid-Containing Materials. US. Patent 4,466,923, 1984.