Esterification reactions catalyzed by Chromobacterium viscosum lipase in CTAB-based microemulsion systems

Chromobacterium viscosum (CV) lipase solubilized in water-in-oil (w/o) microemulsions based on the cationic surfactant hexadecyltrimethylammonium bromide (CTAB) have been used for multigram-scale ester synthesis, including the kinetic resolution of a secondary alcohol. The stability of CV lipase in all the CTAB microemulsions studied was excellent and was superior to that observed in aqueous buffer at the same pH and temperature. Kinetic studies were performed using the synthesis of ethylhexadecanoate as a model reaction. Under pseudo-first-order conditions, the synthesis rates were linearlydependent on the enzyme and fatty acid concentrations and the R dependence shows the characteristic bell-shaped curve (where R = [H(2)O]/[surfactant]). The dependence of enzyme activity toward octyldecanoate synthesis on the pH of the dispersed buffer phase is in marked contrast to that observed for the pH dependence of CV lipase toward p-nitrophenylbutyrate hydrolysis. In the former case, the pH-activity profile is approximately sigmoidal, which may reflect the ionization state of the fatty acid substrate. In the latter case, the pH dependence is minimal at both R = 10 and R = 50, suggesting the enzyme does not experience a changed pH environment. Inclusion of a pH-sensitive probe molecule into those incubations containing fatty acid clearly demonstrates that the probe molecule experiences a changed environment consistent with that expected for the selected buffer. An in situ Fourier transform nuclear magnetic resonance (FT-NMR) assay has been developed which allows continuous monitoring of the esterification reactions, thereby providing an additional means of determining initial rates. The method may be of general value for lipase assays in microemulsions since it may provide, at the same time, information regarding enzyme regioselectivity. (c) 1995 John Wiley & Sons, Inc.     

Activity and stability of horse-liver alcohol dehydrogenase in sodium dioctylsulfosuccinate/cyclohexane reverse micelles

Horse liver alcohol dehydrogenase (EC 1.1.1.1) solubilized in sodium dioctylsulfosuccinate (AOT)/cyclohexane reverse micelles was used for the oxidation of ethanol and reduction of cyclohexanone in a coupled substrate/coenzyme recycling system. The activity of the enzyme was studied as a function of pH and water content. The enzyme was optimally active in microemulsions prepared with buffer of pH around 8. An increase in enzymatic activity was observed as a function of increasing water content. The Km values for the substrates were calculated based on the total reaction volume. The apparent Km for ethanol in reverse micelles was about eight times lower as compared to that in buffer solution, whereas the Km for cyclohexanone was almost unaltered. Storage and operational stability were investigated. It was found that the specific activity of the alcohol dehydrogenase operating in reverse micellar solution was good for at least two weeks. The steroid eticholan-3 beta-ol-17-one was also used as a substrate. In this case the reaction rate was approximately five times higher in a reverse micellar solution than in buffer.     

Properties and repeated use of a reversibly soluble-insoluble yeast lytic enzyme

Summary A yeast lytic enzyme was covalently immobilized on an enteric coating polymer, Eudragit S, that is reversibly soluble and insoluble (S-IS) depending on the pH of the reaction medium. The yeast lytic enzyme immobilized on Eudragit S (Y-E) showed a sharp response of solubility to slight changes in pH without decrease in enzymatic activity. The specific activity per amount of enzyme protein of Y-E for dry yeast cells was about two-thirds that of the native enzyme. In both lysis reactions of dry and pressed baker's yeast cells, changing the pH of the reaction medium from 7.0 to 4.8 at an appropriate interval allows the insoluble Y-E and the reaction products (soluble protein for dry yeast cells and invertase and soluble protein for pressed baker's yeast cells) to be repeatedly separated. The reaction method using a reversible S-IS enzyme is a promising procedure for repeated use of the enzyme in a heterogeneous reaction system containing yeast cells as a substrate.     

Enzymatic synthesis of glucuronides using lipophilic hollow fiber membranes

A lipophilic hollow fiber membrane preparation was used for the enzymatic glucuronidation of lipophilic aromatic compounds. A crude solubilized microsomal enzyme preparation was circulated on the external side of the lipophilic membrane while the phenol containing buffer solution was circulated through the internal side of the hollow fiber membrane. Phenols, which accumulate in and penetrate the lipophilic membrane, were converted by UDP-glucuronyltransferase to the corresponding glucuronides. During this process the lipophilic compounds are converted to hydrophilic substances, which are not able to rediffuse through the lipophilic membrane into the donor side of the hollow fiber module. The produced glucuronide is separated by means of a coupled dialysis with a module of hydrophilic surface (cellulose acetate), while the enzyme protein is retained.On the stripping side of the dialysing module the glucuronide can be separated by solid phase extraction (Lichroprep RP-18) while a continuous substitution of cofactor into this compartment is possible. UDPGA follows its own concentration gradient and migrates into the enzymatic mixture, where it is utilized. This new technique using hollow fiber modules offers completely new possibilities for long-term high-capacity, highly specific glucuronidation of phenolic compounds. Fields of application are not only the economical production of special glucuronides, but also the specific elimination of phenols from waste fluids or from serum and blood of patients.For the production of glucuronides by this technique the use of highly purified enzymes is not essential. Cheap crude enzyme preparations are quite adequate for an optimal reaction. Using a crude enzyme preparation with a specific batch activity of 13 nMol/min per mg of protein, the activity in the reactor system was observed to be 4.6 nMol/min of 2-naphtol glucuronide formed per mg of protein. This corresponds to 3.6 nMol/h of product formed per mg of protein per cm² of hollow fiber surface.Using a membrane surface of 0.5 m² the production of 18 mMol of glucuronide per h and mg protein can be achieved.     

Enzymatic synthesis of arginine-based cationic surfactants

A novel enzymatic approach for the synthesis of arginine N-alkyl amide and ester derivatives is reported. Papain deposited onto solid support materials was used as catalyst for the amide and ester bond formation between Z-Arg-OMe and various long-chain alkyl amines and alcohols (H2N-Cn2, HO-Cn; n = 8-16) in organic media. Changes in enzymatic activity and product yield were studied for the following variables: organic solvent, aqueous buffer content, support for the enzyme deposition, presence of additives, enzyme loading, substrate concentration, and reaction temperature. The best yields (81-89%) of arginine N-alkyl amide derivatives were obtained at 25 degrees C in acetonitrile with an aqueous buffer content ranging from 0 to 1% (v/v) depending on the substrate concentration. The synthesis of arginine alkyl ester derivatives was carried out in solvent-free systems at 50 or 65 degrees C depending on the fatty alcohol chain length. In this case, product yields ranging from 86 to 89% were obtained with a molar ratio Z-Arg-OMe/fatty alcohol of 0.01. Papain deposited onto polyamide gave, in all cases, both the highest enzymatic activities and yields. Under the best reaction conditions the syntheses were scaled up to the production of 2 g of final product. The overall yields, which include reaction, Nalpha-benzyloxycarbonyl group (Z) deprotection and purification, varied from 53 to 77% of pure (99.9% by HPLC) product.     

Thermodynamically based solvent design for enzymatic saccharide acylation with hydroxycinnamic acids in non-conventional media

Enzyme-catalyzed synthesis has been widely studied with lipases (EC 3.1.1.3), but feruloyl esterases (FAEs; EC 3.1.1.73) may provide advantages such as higher substrate affinity and regioselectivity in the synthesis of hydroxycinnamate saccharide esters. These compounds are interesting because of their amphiphilicity and antioxidative potential. Synthetic reactions using mono- or disaccharides as one of the substrates may moreover direct new routes for biomass upgrading in the biorefinery. The paper reviews the available data for enzymatic hydroxycinnamate saccharide ester synthesis in organic solvent systems as well as other enzymatic hydroxycinnamate acylations in ionic liquid systems. The choice of solvent system is highly decisive for enzyme stability, selectivity, and reaction yields in these synthesis reactions. To increase the understanding of the reaction environment and to facilitate solvent screening as a crucial part of the reaction design, the review explores the use of activity coefficient models for describing these systems and - more importantly - the use of group contribution model UNIFAC and quantum chemistry based COSMO-RS for thermodynamic predictions and preliminary solvent screening. Surfactant-free microemulsions of a hydrocarbon, a polar alcohol, and water are interesting solvent systems because they accommodate different substrate and product solubilities and maintain enzyme stability. Ionic liquids may provide advantages as solvents in terms of increased substrate and product solubility, higher reactivity and selectivity, as well as tunable physicochemical properties, but their design should be carefully considered in relation to enzyme stability. The treatise shows that thermodynamic modeling tools for solvent design provide a new toolbox to design enzyme-catalyzed synthetic reactions from biomass sources.     

脂肪酶在微乳液和微乳液凝胶中催化辛酸辛醇的酯化反应

脂肪酶在合成反应中具有很高的区域选择性和立体选择性 ,已广泛用于食品工业和药物工业[1,2 ] ,在有机介质中的脂肪酶催化反应已有较多研究[3 ,4 ] 。微乳液一般由表面活性剂、助表面活性剂、油和水等组份组成 ,它是一种热力学稳定、光学透明、宏观均匀而微观不均匀的体系 ,能提供酶催化所需要的巨大油 /水界面[5] 。而将脂肪酶增溶于油包水(Ｗ /Ｏ)微乳液中的纳米级“水池”中 ,可使酶以分子水平分散[6] ,图 1(ａ) ,从而可用来模拟细胞微环境中的反应。油包水微乳液中的酶可通过加入明胶而制成固定化酶 ,含明胶的微乳液凝胶 (ＭＢＧｓ)最早…     

Purification and characterization of guanosine diphospho-D-mannose dehydrogenase. A key enzyme in the biosynthesis of alginate by Pseudomonas aeruginosa

Alginate-producing Pseudomonas aeruginosa are usually associated with the cystic fibrosis lung environment and contribute to the high mortality rates observed among these patients. The present paper describes the purification and enzymatic properties of guanosine diphospho-D-mannose dehydrogenase (EC 1.1.1.132), a key enzyme in alginate biosynthesis by mucoid P. aeruginosa. The enzyme was overproduced using a plasmid vector containing algD (the gene encoding this enzyme) under control of the tac promoter. It was purified from cell-free lysates by lowering the pH to 5.0, heating the extract to 57.5 degrees C for 10 min, and discarding the protein pellet. The enzyme was selectively precipitated from the supernatant fraction with 45% acetone, resuspended in a 100 mM triethanolamine acetate buffer, pH 7.6, and ultimately purified by Bio-Sil TSK-400 gel filtration chromatography. The subunit molecular weight (Mr 48,000) as well as the N-terminal amino acid sequence corresponded to those predicted from the DNA sequence of algD. The native protein migrated as a hexamer of 290,000 molecular weight upon Bio-Gel A-1.5m gel filtration chromatography. Kinetic analysis demonstrated an apparent Km of 14.9 microM for the substrate GDP-D-mannose and 185 microM for the cofactor NAD+. GDP-D-mannuronic acid was identified as the enzyme reaction product. Several compounds (including GMP, ATP, GDP-D-glucose, and maltose) were found to inhibit enzymatic activity. GMP, the most potent of these inhibitors, exhibited competitive inhibition with an apparent Ki of 22.7 microM. Enzyme activity was also sensitive to the sulfhydryl group modifying agents iodoacetamide and p-hydroxymercuribenzoate. The addition of excess dithiothreitol restored enzyme activity, suggesting a possible involvement of cysteine residues in enzymatic activity.     

A kinetic model for enzyme interfacial activity and stability: pa-hydroxynitrile lyase at the diisopropyl ether/water interface

A kinetic framework is developed to describe enzyme activity and stability in two-phase liquid-liquid systems. In particular, the model is applied to the enzymatic production of benzaldehyde from mandelonitrile by Prunus amygdalus hydroxynitrile lyase (pa-Hnl) adsorbed at the diisopropyl ether (DIPE)/aqueous buffer interface (pH = 5.5). We quantitatively describe our previously obtained experimental kinetic results (Hickel et al., 1999; 2001), and we successfully account for the aqueous-phase enzyme concentration dependence of product formation rates and the observed reaction rates at early times. Multilayer growth explains the early time reversibility of enzyme adsorption at the DIPE/buffer interface observed by both enzyme-activity and dynamic-interfacial-tension washout experiments that replace the aqueous enzyme solution with a buffer solution. The postulated explanation for the unusual stability of pa-Hnl adsorbed at the DIPE/buffer interface is attributed to a two-layer adsorption mechanism. In the first layer, slow conformational change from the native state leads to irreversible attachment and partial loss of catalytic activity. In the second layer, pa-Hnl is reversibly adsorbed without loss in catalytic activity. The measured catalytic activity is the combined effect of the deactivation kinetics of the first layer and of the adsorption kinetics of each layer. For the specific case of pa-Hnl adsorbed at the DIPE/buffer interface, this combined effect is nearly constant for several hours resulting in no apparent loss of catalytic activity. Our proposed kinetic model can be extended to other interfacially active enzymes and other organic solvents. Finally, we indicate how interfacial-tension lag times provide a powerful tool for rational solvent selection and enzyme engineering.     

The enantioselective hydrolysis of 3-hydroxy-5-phenyl-4-pentenoicacidethylester in supercritical carbon dioxide using lipases

A new experimental high-pressure-unit was constructed for the enantioselective enzymatic hydrolysis of 3-hydroxy-5-phenyl-4-pentenoicacidethylester (a precursor for biological interesting substances) in a biphasic buffer/SCCO(2)-system. One objective is to take advantage of the solubility differences of the substrate and the produced acid. Thus the different solubilities of the substrates and the products in the different phases were studied regarding to an overall process integration. One ester enantiomer is preferably hydrolyzed, the other remains in the supercritical phase. And the produced acid enantiomer is concentrated in the buffer phase. The decrease in pressure is followed by an extraction process of the remaining substrate-enantiomer, in consequence it will be possible to combine an enzymatic reaction with a separation step. The catalysis was optimized in regard to enantioselectivity, enantiomeric excess, conversion and reaction time. A high enantioselectivity is achieved for the aromatic substrate using the lipase of Pseudomonas cepacia. The results show that this unconventional reaction system offers tremendous advantages for enzyme process development.     

Reaction of triosephosphate isomerase with L-glyceraldehyde 3-phosphate and triose 1,2-enediol 3-phosphate

Triosephosphate isomerase catalyzes the isomerization and/or racemization reactions of L-glyceraldehyde 3-phosphate (LGAP), the enantiomer of the physiological substrate. The reaction is inhibited by the active site directed reagent glycidol phosphate. The amount of protonation product formation catalyzed by a fixed enzyme concentration is nearly independent of increasing steady-state concentrations of triose 1,2-enediol 3-phosphate caused by buffer catalysis of LGAP deprotonation. Therefore, enzymatic protonation of the enediol or enediolate, which could account for the observed enzymatic catalysis of LGAP isomerization and/or racemization, is at best a minor reaction. Instead LGAP reacts directly at the enzyme active site. Triosephosphate isomerase catalysis of the protonation of triose 1,2-enediol 3-phosphate was expected because of the strong evidence supporting an enediol reaction intermediate for the overall reaction catalyzed by isomerase. The most reasonable explanation for the failure to observe enzymatic protonation is that in solution the enediol undergoes beta elimination of phosphate (t 1/2 is estimated to be 10(-6) s) faster than it can diffuse to and form a complex with isomerase.     

Enzymatic conversion of dehydrodivanillin to vanillin by an anaerobic recombinant FE7

Enzymatic degradation of dehydrodivanillin (DDV) was studied using high performance liquid chromatography (HPLC) with an anaerobic DDV-degrading recombinant FE7 under both aerobic and anaerobic conditions. When 200 mg of FE7 cells were mixed with 40 μg DDV in 1 ml phosphate buffer (0.01 M, pH 7.0) and 10 mM mercaptoethanol and incubated at 37°C for 24 h under an O₂-free CO₂ atmosphere, about 20 μg of DDV was decomposed. Only 12 μg DDV could be degraded when the same reaction was done under aerobic conditions, suggesting that the reaction occurs more easily under anaerobic than aerobic conditions. Enzymatic degradation of DDV was performed using a cell-free extract as a crude enzyme solution under aerobic conditions in a similar way. A reaction product detected and analysed by thin layer, high performance liquid and gas chromatographies and mass spectrometry was found to be vanillin from enzymatic reaction mixture. This enzymatic activity was not detected in either the culture supernatant or the heat-inactivated control. These results suggest that there may be an intracellular enzyme system which is involved in the conversion of DDV to vanillin. This is the first report to study the enzymatic degradation of DDV by anaerobes.     

Kinetic modeling of enzymatic hydrolysis of cellulose in differently pretreated fibers from dairy manure

A kinetic model incorporating dynamic adsorption, enzymatic hydrolysis, and product inhibition was developed for enzymatic hydrolysis of differently pretreated fibers from a nitrogen-rich lignocellulosic material-dairy manure. The effects of manure proteins on the enzyme adsorption profile during hydrolysis have been discussed. Enzyme activity, instead of protein concentration, was used to describe the enzymatic hydrolysis in order to avoid the effect of manure protein on enzyme protein analysis. Dynamic enzyme adsorption was modeled based on a Langmiur-type isotherm. A first-order reaction was applied to model the hydrolysis with consideration being given for the product inhibition. The model satisfactorily predicted the behaviors of enzyme adsorption, hydrolysis, and product inhibition for all five sample manure fibers. The reaction conditions were the substrate concentrations of 10-50 g/L, enzyme loadings of 7-150 FPU/g total substrate, and the reaction temperature of 50 degrees C.     

In Situ Hydration of Enzymes in Non-Polar Organic Media Can Increase the Catalytic Rate

The nature of the buffer species used in the drying process is important when lyophilized enzyme preparations are suspended in organic media. The activity of subtilisin Carlsberg in a transesterification reaction was found to vary depending on the nature of the buffer used. It was postulated that the large excess of salt present in the dried powder could be affecting enzymatic activity by alterations to the microscopic structure of the powder. To establish if this were true, microscopic changes were eliminated by covalently immobilising the enzyme onto a macroporous polymer support so that the counter-ions could be exchanged by washing with dilute salt solutions. It was found that in the immobilised samples no significant effects of salt ions were noted. This was the case even when salt ions were in considerable excess of that needed to balance protein charges. Hence the activity variations noted in freeze-dried powders are probably due to changes to the microscopic structure, rather than to molecular scale interactions. Similarly the previously observed activating effect of crown ether solutions on freeze-dried powders is not repeated on an immobilised preparation suggesting that this too may be due to a microscopic effect on the powder.     

Enzymes and microemulsions. Activity and kinetic properties of liver alcohol dehydrogenase in ionic water-in-oil microemulsions

The activity and the kinetic properties of horse liver alcohol dehydrogenase have been studied in water-in-oil microemulsions containing sodium dodecyl sulfate (SDS) or hexadecyl trimethylammonium bromide (CTAB), 1-butanol or 1-pentanol or 1-hexanol or t-butanol, water and cyclohexane alone or with octane. In the anionic microemulsions (i.e. containing sodium dodecyl sulfate), the enzyme quickly lost its activity, but was efficiently protected by the coenzyme and some adenine nucleotides. In the cationic microemulsions (i.e. containing hexadecyl trimethylammonium bromide), the enzyme activity was more stable and with higher alcohols was stable for at least 20 min. The Michaelis constant of NAD+ calculated with respect to the water content was nearly constant and higher than in water. The maximum velocity in anionic microemulsions depends on the water content whereas in cationic microemulsions, the maximum velocity did not show a clear dependence on the water content and was close to the maximum velocity found in water. The pH dependence of Km and Vmax in these microemulsions was similar to that observed in water. The kinetic data for a hydrophobic substrate, cinnamyl alcohol, showed that this alcohol partitions between the pseudo-phases and thus the apparent Michaelis constant and the concentration at which substrate-excess inhibition appeared were increased. The catalytic properties of the enzyme in microemulsions were illustrated by the preparative reduction of cinnamaldehyde with cofactor recycling. The rate determination of NAD+ reduction and of 1-butanol/cinnamaldehyde redox reaction showed that at low water content (2.8%), the NAD+ reduction rate was close to zero whereas the redox reaction rate was about half of the rate at higher water content. Probably at low water content the coenzyme binding-dissociation rates are reduced much more than the binding-dissociation rates of the substrates and the rates of the ternary complex interconversion. The cationic microemulsions seemed to be very favorable medium for enzyme activity, the tetraalkyl ammonium surfactant causing less denaturation than the anionic detergent dodecyl sulfate.     

A new approach to preparative enzymatic synthesis

A new approach to preparative organic synthesis in aqueous–organic systems is suggested. It is based on the idea that the enzymatic process is carried out in a biphasic system “water–water-immiscible organic solvent.” Thereby the enzyme is localized in the aqueous phase—this eliminates the traditional problem of stabilizing the enzyme against inactivation by a nonaqueous solvent. Hence, in contrast to the commonly used combinations “water–water-miscible organic solvent,” in the suggested system the content of water may be infinitely low. This allows one to dramatically shift the equilibrium of the reactions forming water as a reaction product (synthesis of esters and amides, polymerization of amino acids, sugars and nucleotides, dehydration reactions, etc.) toward the products. The fact that the system consists of two phases provides another very important source for an equilibrium shift, i.e., free energies of the transfer of a reagent from one phase to the other. Equations are derived describing the dependence of the equilibrium constant in a biphasic system on the ratio of the volumes of the aqueous and nonaqueous phases and the partition coefficients of the reagents between the phases. The approach has been experimentally verified with the synthesis of N-acetyl-L -tryptophan ethyl ester from the respective alcohol and acid. Porous glass was impregnated with aqueous buffer solution of chymotrypsin and suspended in chloroform containing N-acetyl-L -tryptophan and ethanol. In water (no organic phase) the yield of the ester is about 0.01%, whereas in this biphasic system it is practically 100%. The idea is applicable to a great number of preparative enzymatic reactions.     

Immobilized α-chymotrypsin: Pore diffusion control owing to pH gradients in the catalyst particles

The fast enzymatic hydrolysis of D ,L -phenylalanine methylester (DLE) to L -phenylalanine (LA) and D -phenylalanine methylester (DE) with immobilized α-chymotrypsin was chosen as a model reaction. Under the experimental conditions applied in the present investigations the pore diffusion is the rate-limiting step of this reaction owing to the pH gradient in the particles. The effectiveness of the catalyst is experimentally determined as a function of the substrate concentration based on measurements of the enzyme protein content of native and immobilized enzyme. The proteolytic reaction is theoretically treated by also using a pore diffusion model which takes into account the concentration gradients of substrate and product, pH- and enzyme activity profiles, as well as the change of buffer capacity of the solute in the catalyst particles. The model parameters were experimentally determined for the investigated system. It can be shown that conditions are possible for which the effectiveness of the catalyst exceeds unity.     

In vitro activation of a soluble cholesterol esterase from bovine adrenals by a cAMP-dependent protein kinase.

Properties and partial purification of the bovine adrenal cholesterol esterase from the 100000 X g supernatant fraction were investigated. Variations of the enzyme activity with time-dependent (enzymatic) and time-dependent (non enzymatic) effects have been demonstrated. Mg2 has been proved to inhibit the enzyme activity by a non-enzymatic effect in 50mM Tris/HCl buffer, pH 7.4. A time-dependent inactivation of the cholesterol esterase has been observed in the same buffer. The enzyme could be protected from this enzymatic inactivation by its substrate, cholesterol oleate. cAMP, ATP and Mg2 cuase a time-dependent stimulation of the enzyme in 50mM Tris/HCl buffer, pH 7.4. This result suggests that corticotropin activates the soluble cholesterol esterase from bovine adrenals via cAMP-dependent protein kinase. This view is strengthened by the incorporation of 32P radioactivity from [gamma-32P] ATP into the protein fraction of the 100,000 X g supernatant. The protein-bound 32P radioactivity could be co-purified with the enzyme activity during the partial purification of the soluble cholesterol esterase.     

Directed evolution of an extremely fast phosphotriesterase by in vitro compartmentalization

We describe the selection of a phosphotriesterase with a very fast k(cat) (over 10(5) s(-1)), 63 times higher than the already very efficient wild-type enzyme. The enzyme was selected from a library of 3.4 x 10(7) mutated phosphotriesterase genes using a novel strategy based on linking genotype and phenotype by in vitro compartmentalization (IVC) using water-in-oil emulsions. First, microbeads, each displaying a single gene and multiple copies of the encoded protein, are formed by compartmentalized in vitro translation. These microbeads can then be selected for catalysis or binding. To select for catalysis the microbeads are re-emulsified in a reaction buffer of choice with a soluble substrate. The product and any unreacted substrate are coupled to the beads when the reaction is finished. Product-coated beads, displaying active enzymes and the genes that encode them, are detected with anti-product antibodies and selected using flow cytometry. This completely in vitro process selects for all enzymatic features simultaneously (substrate recognition, product formation, rate acceleration and turnover) and single enzyme molecules can be detected.     

A rapid assay for activity of phospholipase A2 using radioactive substrate

A rapid method for the assay of phospholipase A2 has been developed using a radioactive substrate, L-alpha-dipalmitoyl-(2-[9,10(N)-3H]palmitoyl)-phosphatidylcholine. The substrate diluted with cold carrier (1 mM) is dissolved in 80% ethanol containing 25 mM sodium deoxycholate. The enzymatic reaction is performed in 1.0 ml 0.1 M glycine-NaOH buffer, pH 9.0, containing 2 mumol CaCl2, 10 micrograms bovine serum albumin, 2.5 mumol sodium deoxycholate, 0.01 unit (or less) phospholipase A2, and 40-100 nmol substrate. The enzymatic reaction is terminated by adding 0.2 ml 5% Triton X-100 solution containing 40 mumol EDTA. The product of the enzymatic reaction, radioactive palmitic acid, is extracted by 10 ml hexane containing 0.1% acetic acid in the presence of anhydrous sodium sulfate (0.5 g/ml). Activity of phospholipase A2 is directly determined from the radioactivity in the hexane extract. The present method achieves a quick separation of the radioactive product, [3H]palmitic acid, from the radioactive substrate, L-alpha-dipalmitoyl-(2-[3H]palmitoyl)-phosphatidylcholine, without the need of separation by TLC.