Influence of microwave irradiation on enzymatic properties: applications in enzyme chemistry

Although microwave-assisted reactions are widely applied in various domains of organic chemistry, their use in the area of enzyme chemistry has been rather limited, due to the high temperatures associated with the microwave heating: Because current technology, allows a good control of reaction parameters, several examples of microwave-assisted enzyme chemistry have been reported, using stable and effective biocatalysts (modified enzymes). The purpose of this review is to highlight the applications and studies on the influence of microwave irradiation on enzymatic properties and their application in enzyme chemistry.     

Effect of microwave irradiation on the physical properties and morphological structures of cotton cellulose

Microwave heating has been proved to be more rapid, uniform and efficient, and easily penetrate to particle inside. To investigate the effect of microwave irradiation on the physical property and morphological structure of cotton cellulose, cellulose fabric was treated with microwave irradiation at different conditions. The physical properties of the treated cellulose fabric were investigated. The morphological structures and thermal stabilities of the untreated and treated cellulose were investigated with differential scanning calorimetry (DSC) and X-ray diffraction. The results show that the physical properties of the treated cellulose fabrics were improved and the recoverability had not significant change. The thermal stability of the treated cellulose was changed. The crystallinity and preferred orientation of the treated cotton cellulose increased.     

Effect of microwave irradiation on the molecular and structural properties of hyaluronan

Hyaluronan (Na⁺ salt of hyaluronic acid, HA) was extensively depolymerised by HCl-catalyzed hydrolysis at pH 3 for up to 500 min under temperature-controlled microwave irradiation. The effects of microwave heating on the hydrodynamic properties of the polysaccharide were determined by SEC-MALLS and viscometry. The weight-average molecular mass (M_w) of HA decreased from 1.44 × 10⁶ to 5000, reaching the region of higher oligosaccharides. The scission of HA chains was found to proceed randomly during the whole degradation process. Treatment of the M_w and intrinsic viscosity data according to the Mark–Houwink equation, [η] = k × M_w^α suggested three relationships with α₁ = 0.46 for M_w > 500,000, α₂ = 0.84 for M_w between 500,000 and 50,000, and α₃ = 1.13 for M_w < 50,000. The results revealed that HA with M_w > 10,000 adopts a stiffish coil conformation in solution. As monitored by FT-IR and NMR spectroscopic techniques, the primary structure of the HA chains was maintained during the microwave-assisted hydrolysis at pH 3 at 105 °C. At reaction times larger than 240 min, uv spectroscopy suggested the depolymerisation of HA was accompanied by formation of by-products produced by side reaction.     

Structure and properties of starch-based foams prepared by microwave heating from extruded pellets

The physical properties of microwave-foamed starch-based pellets, including density, porosity, cell structure, water absorption characteristics and mechanical properties were characterized. It was found that the physical properties of these starch-based foams produced by microwave heating are highly dependent on the raw materials and additives. Foam density decreased significantly after addition of 5.5–10.5% w/w salts, while foams containing nucleation agent (talc) were denser than the control with reduced cell size. A proprietary blowing agent did not affect the foam density markedly. Addition of salts also increased the water sorption of foams and plasticized cell walls. Mechanical behaviour of foamed pellets can be adjusted effectively by controlling the cell structure through using different additives. Mechanical properties of the foamed pellets in the elastic region as well as under large deformation (up to 40% strain) all follow a power–law relationship with foam density.     

Impact of microwave heating on the physico-chemical properties of a starch–water model system

The objective of this work was to understand the physico-chemical changes induced in a wheat starch model system as a result of microwave heating. Wheat starch dispersions in water, with final solids content of 33%, 40% or 50%, were heated in a microwave oven. Following heating the samples were stored at 25 °C for up to 120 h and analyzed periodically. Microwave heated gels were significantly different from conduction heated gels in all parameters measured. The differences in properties are a reflection of the differences in the heat and mass transfer of the different modes of heating. The lack of granule swelling and the resulting soft gel are two key observations. The results of this study suggest a different mechanism of starch gelatinization compared to conduction heating. The vibrational motion and the rapid increase in temperature also result in granule rupture and formation of film polymers coating the granule surface.     

Preparation of carboxymethyl chitosan in aqueous solution under microwave irradiation

Carboxymethyl chitosan was prepared by reacting chitosan with chloroacetic acid in water under microwave irradiation. The effect of the reaction conditions was investigated and optimal conditions were identified. The influence of mass ratio of chloroacetic acid to chitosan, microwave power and pH on the degree of substitution or intrinsic viscosity were further studied. The degree of substitution of the carboxymethyl chitosan synthesized exceeded 0.85.     

Electrochemical investigation of immobilized hemoglobin: redox chemistry and enzymatic catalysis

Hb entrapped in the Konjak glucomannan (KGM) film could transfer electrons directly to an edge-plane pyrolytic graphite (EPG) electrode, corresponding to the redox couple of Fe(III)/Fe(II). The redox properties of Hb, such as formal potential, electron transfer rate constant, the stability of the redox state of protein and redox Bohr effect, were characterized by cyclic voltammetry and square wave voltammetry. The stable Hb-KGM/EPG gave analytically useful electrochemical catalytic responses to oxygen, hydrogen peroxide and nitrite.     

Influence of the immobilization chemistry on the properties of immobilized β-galactosidases

Neutral β-galactosidases (from E. coli and K. lactis) were bound to glutaraldehyde-agarose (Glut-agarose) through amino groups, and to thiolsulfinate-agarose (TSI-agarose) through thiol groups. In general, TSI-gels exhibited higher yields after immobilization (60–85%) than Glut-gels (36–40%). The kinetic parameters of the enzymes bound to TSI-gels (particularly those with lower concentration of active groups) were less affected than those of the Glut-gels. This might indicate that the binding to TSI-agarose is more conservative of the protein conformation. However, the Glut-derivatives exhibited in general better thermal and solvent stabilities than TSI-derivatives. The stability of the derivatives was studied in the presence of ethanol, dioxane and acetone (18% v/v). The stabilization of the immobilized enzymes, for some of the solvents assayed, was evidenced by the existence of final very stable enzyme states with high residual activities, thus allowing the utilization of the derivatives in the presence of organic cosolvents.     

Series-type enzyme deactivations: Influence of intermediate activity on deactivation kinetics

A series-type enzyme deactivation model involving an active enzyme precursor is proposed wherein the enzyme activity is a weighted function of the active enzyme states. The active enzyme precursor may be less active, as active or more active than the initial enzyme form. The proposed model is shown to fit the soluble and immobilized enzyme deactivation data presented reasonably well. Some enzymes exhibit a ‘compensation-like’ effect. In other enzymes, if the deactivation rate coefficient for the second step, k₂, is zero, then the activity may stabilize to a value that depends upon the relative activities of the two active enzyme states.     

Degradation of hyaluronan by ultrasonication in comparison to microwave and conventional heating

Hyaluronan (hyaluronic acid, HA) was depolymerised by ultrasonication (US), microwave irradiation (MW) and conventional heating (CH), and the effect of pH and oxidants was investigated. The degradation was followed by viscometry and size exclusion chromatography coupled with low-angle light scattering. The results demonstrated that depolymerisation of HA by US leveled off to a limiting molecular mass, and the degradation was significantly enhanced by acidic and alkaline pH only in the presence of oxidants. In contrast to US, the course of depolymerisation by MW was strongly pH-dependent, and the degradation rate increased with decreasing pH. The expected enhancement of depolymerisation by MW in comparison to CH was marked only at very short heating time at pH <4. The NMR and FTIR spectral analyses indicated that HA in the whole M_w-range studied retained almost the backbone of the parent polysaccharide independently on the degradation method used. At harsh degradation conditions (long-term treatments, particularly at acidic pH or alkaline pH and in presence of oxidants) the depolymerisation was accompanied by destruction of both constituent sugar residues and formation of unsaturated structures detectable by UV-absorption at 230–240 and 260–270 nm. US-assisted oxidative degradation under mild reaction conditions was shown to be the most appropriate procedure to reduce the molecular mass of HA to 100 kDa without significant chemical modification of the polysaccharide.     

Applications of microwave dielectric heating in environment-related heterogeneous gas-phase catalytic systems

The application of microwave dielectric heating in a range of environment-related heterogeneous catalytic reaction systems has been reviewed. The reactions investigated include the decomposition of hydrogen sulfide, the reduction of sulfur dioxide with methane, the reformation of methane by carbon dioxide, the hydrodesulfurization of thiophene, and the oxidative coupling of methane. The interaction of microwave irradiation with heterogeneous catalytic systems and its consequence for the microwave heating behaviour of catalysts have been examined. The effect/mechanism of microwave dielectric heating on heterogeneous catalytic reaction systems has also been discussed.     

Comparative study on microwave and conventional methods for protein hydrolysis in food

Summary A rapid microwave hydrolysis procedure was developed for amino acid determination in food. The hydrolysis was performed with 6M HCl in sealed vessels using a microwave digestion system.Bovine Serum Albumin was chosen as a model protein to compare its theoric amino acid sequence with the experimental results obtained after hydrolysis by both the traditional oven heating and the microwave methods. Furthermore the selected microwave methods were carried out on different food matrices (cheese and durum wheat) and the obtained data were compared with the traditional method results.This comparative study shows that the rapid microwave hydrolysis is as accurate and precise as the traditional hydrolysis for determining amino acids in food.These results were presented at the Third International Congress on Amino Acids, Vienna, August 23–27th, 1993.     

Changes of amino acid gradients in brain tissues induced by microwave irradiation and other means

Focused microwave irradiation to the head (FMI) has been used extensively by neurochemists for rapid inactivation of enzymatic activity in brain tissues and the preservation, for in vitro analysis, of in vivo substrate concentrations. Periodically the suitability of this technique for regional studies has been questioned. Evidence has now been obtained, on the basis of altered concentration gradients for GABA and taurine from the Substantia Nigra (SN) to an Adjacent Dorsal Area (ADJ), that FMI not only inactivates enzymes, but also facilitates rapid diffusion of small molecules from areas of high concentrations to adjacent areas of lower concentration. To a lasser extent, the implantation of plastic injection cannulas also decreased these concentration gradients. These results offer clear evidence that FMI is ill suited and unreliable for studies designed to map and compare the in vivo regional concentrations of diffusible organic molecules (such as amino acids) in brain tissues. Any invasive technique that compromises membrane barriers is likely to produce smaller similar effects.     

Effect of support material and enzyme pretreatment on enantioselectivity of immobilized subtilisin in organic solvents

Subtilisin Carlsberg was covalently attached to five macroporous acrylic supports of varying aquaphilicity (a measure of hydrophilicity). Kinetic parameters of the transesterification of S and R enantiomers of secphenethyl alcohol with vinyl butyrate, catalyzed by various immobilized subtilisins, were determined in anhydrous dioxane and acetonitrile. Enzyme enantioselectivity in acetonitrile, but not in dioxane, correlated with the aquaphilicity of the support; a mechanistic rationale for this phenomenon was proposed. Although the catalytic activity of immobilized subtilisin in anhydrous solvents strongly depended on enzyme pretreatment, the enantioselectivity was essential conserved. (c) 1994 John Wiley & Sons, Inc.     

Thin-layer liquid crystal thermometry of cells in vitro during hyperthermal microwave irradiation

A nonperturbing technique of thin-layer liquid crystal thermometry was developed to quantitate heating of Chinese hamster ovary cells and the bacterium Serratia marcescens when exposed to 2450-MHz microwave fields at 0.2–0.5 W/cm². Cells suspended in culture medium were injected into 5-cm glass microcapillary tubes coated on the inside with a thin layer of liquid crystal. The tubes were sealed and placed parallel to the electric field in a watertight waveguide exposure chamber where they were heated by circulating temperature-controlled water. Even at high circulation rates, liquid crystal color changes indicated local microwave capillary tube heating of 0.1–0.25 °C. Precision of measurement was 0.02 °C. Observations during microwave heating were significantly different from observations without microwaves at the 1% level, and heating increased as circulating water flow was reduced from 300 ml/s to 100 ml/s. The results of a cell survival assay following hyperthermal treatment were in good agreement with expectations based on the observations of microwave heating using liquid crystals.     

Ionic strength: A neglected variable in enzyme technology

Ionic strength is a variable of considerable importance in enzyme biochemistry. Unfortunately, the ionic strength variable is little used in immobilized enzyme studies. This paper gives examples of enzyme systems known to be sensitive to ionic strength. Suggestions are offered for the manipulation of ionic strength in enzyme technology studies. Laboratory results on the effect of ionic strength on the reaction catalysed by immobilized pancreatic ribonuclease (EC 3.1.27.5) are also presented and analysed using the Debye-Hückel equation.     

Enzymatic synthesis of cetyl oleate in a solvent-free medium using microwave irradiation and physicochemical evaluation

Abstract

A cosmetic ester, cetyl oleate was synthesized using microwave irradiated system. The esterification reaction was carried using Candida antarctica lipase B in a solvent-free media. The influence of various reaction parameters was studied, and the efficiency of Fermase CALB^TM10000 was compared with other enzymes. Equilibrium conversion of 97.5% was obtained within 20?min at 60?°C temperature, 1:2 oleic acid to cetyl alcohol molar ratio and 4% w/w dose of lipase. A comparative study showed that microwave irradiation is a much more efficient method than ultrasound irradiation and conventional heating. Fermase CALB^TM10000 was reusable over 6 enzymatic cycles as its stability improved under microwave system. Physicochemical parameters of cetyl oleate were tested in order to analyze its suitability for further cosmetic use.     

Lipase catalyzed esterification of glycidol in nonaqueous solvents: solvent effects on enzymatic activity

We studied the effect of organic solvents on the kinetics of porcine pancreatic lipase (pp) for the resolution of racemic glycidol through esterification with butyric acid. We quantified ppl hydration by measuring water sorption isotherms for the enzyme in the solvents/mixtures tested. The determination of initial rates as a function of enzyme hydration revealed that the enzyme exhibits maximum apparent activity in the solvents/mixtures at the same water content (9% to 11% w/w) within the associated experimental error. The maximum initial rates are different in all the media and correlate well with the logarithm of the molar solubility of water in the media, higher initial rates being observed in the solvents/mixtures with lower water solubilities. The data for the mixtures indicate that ppl apparent activity responds to bulk property of the solvent. Measurements of enzyme particle sizes in five of the solvents, as function of enzyme hydration, revealed that mean particle sizes increased with enzyme hydration in all the solvents, differences between solvents being more pronounced at enzyme hydration levels close to 10%. At this hydration level, solvents having a higher water content lead to lower reaction rates; these are the solvents where the mean enzyme particle sizes are greater. Calculation of the observable modulus indicates there are no internal diffusion limitations. The observed correlation between changes in initial rates and changes in external surface area of the enzyme particles suggests that interfacial activation of ppl is only effective at the external surface of the particles. Data obtained for the mixtures indicate that ppl enantioselectivity depends on specific solvent-enzyme interactions. We make reference to ppl hydration and activity in supercritical carbon dioxide. (c) 1994 John Wiley & Sons, Inc.     

Influence of the porous texture of silica gels on the enzymatic activity of lipases in esterification reactions

Pseudomonas Cepacia lipases were encapsulated in hybrid silica-polyvinyl alcohol gels, which were dried either supercritically in order to form aerogels or by evaporation so as to obtain xerogels. In each case, the catalytic activity of the encapsulated enzymes was studied and compared to free enzyme biocatalysis. This study demonstrates that the activity of the enzyme is increased when the procedure used allows it to resist capillary stresses occurring during the drying of the gel. That is, esterification rates are higher when the gels are synthesized with a base catalyst, such as NaF, in the presence of polyvinyl alcohol and then dried supercritically.     

Generation of a pH gradient in an immobilized enzyme system

Several examples of two-step sequential reactions exist where, because of the poor equilibrium conversion by the first reaction, it is desirable to conduct the two reactions simultaneously. In such a scheme, the product of the first reaction is continuously removed by the second reaction, thus not allowing the first reaction to approach chemical equilibrium. Therefore, the first reaction is allowed to proceed in the desired direction at an appreciable rate. However, in many biochemical applications where enzyme catalysts are involved, the enzyme's activities are strong functions of pH. Where the pH optima of the first and second reaction differ by three to four units, the above reaction scheme would be difficult to implement. In these cases, the two reactions can be separated by a thin permeable membrane across which the desired pH gradient is maintained. In this article, it was shown, both by theory and experiment, that a thin, flat membrane of immobilized urease can accomplish this goal when one face of the membrane is exposed to the acidic bulk solution (pH(b) = 4.5) containing a small quantity of urea (0.01 M). In this particular case, the ammonia that was produced in the membrane consumed the incoming hydrogen ions and thus maintained the desired pH gradient. Experimental results indicate that with sufficient urease loading, the face of the membrane opposite to the bulk solution could be maintained at a pH that would allow many enzymes to realize their maximum activities ( approximately 7.5). It was also found that this pH gradient could be maintained even in the presence of a buffer, which greatly enhances the transport of protons into the membrane. (c) 1993 John Wiley & Sons, Inc.