Tyrosinase: polybrene noncovalent complexes in water-ethanol mixtures

Formation of noncovalent complexes between tyrosinase from mushrooms and a cationic polyelectrolyte, polybrene (PB, poly (1,5-dimethyl-1,5-diazaundecamethyelene) bromide), was shown to activate and stabilize tyrosinase in water-ethanol mixtures. In the reaction of catechol oxidation in aqueous solutions, catalytic activity (k(cat)) of tyrosinase-PB complex ([PB]/[tyrosinase] molar ratio 100:1, per mole of polymer) in a wide range of pH was higher than that of free tyrosinase. In aqueous solutions and in water-ethanol mixtures at moderate concentrations of ethanol (10-40% v/v), the value of k(cat) of tyrosinase-PB complex exceeded the activity of free enzyme, from 1.2-2-fold, accompanied by the essential (up to 10-fold) increase in the value of the specificity constant (k(cat)/K(m)). The results are of practical importance for the construction of biocatalysts working successfully in polar organic media.     

Addition of negatively charged residues can reverse the decrease in the solubility of an acidic protein caused by an artificially introduced non-polar surface patch

A non-polar patch on the surface of a protein can cause a reduction in the solubility and stability of the protein, and thereby induce aggregation. However, a non-polar patch may be required so that the protein can bind to another molecule. The mutant 6L—derived from the acidic, dimeric α-helical protein sulerythrin and containing six additional leucines arranged to form a non-polar patch on its surface when properly folded—has a substantially reduced solubility in comparison with that of wild-type sulerythrin. This reduced solubility appears to cause 6L to aggregate. To reverse this aggregation, we mutated 6L so that it contained three to six additional glutamates or aspartates that we predicted would surround the non-polar leucine patch on natively folded 6L. Although the introduction of three glutamates or aspartates increased solubility, the mutants still aggregate and have a reduced α-helical content. Conversely, mutants with six additional glutamates or aspartates appear to exist mostly as dimers and to have the same α-helical content as that of wild-type sulerythrin. Notably, the introduction of five lysines or five arginines at the positions held by the glutamates or aspartates did not recover solubility as effectively as did the negatively charged residues. These results demonstrate that negatively charged residues, but not positively charged ones, surrounding a non-polar patch on an acidic protein can completely reverse the decrease in its solubility caused by the patch of non-polar surface residues.     

Computer-assisted mass spectrometric analysis of naturally occurring and artificially introduced cross-links in proteins and protein complexes

A versatile software tool, VIRTUALMSLAB, is presented that can perform advanced complex virtual proteomic experiments with mass spectrometric analyses to assist in the characterization of proteins. The virtual experimental results allow rapid, flexible and convenient exploration of sample preparation strategies and are used to generate MS reference databases that can be matched with the real MS data obtained from the equivalent real experiments. Matches between virtual and acquired data reveal the identity and nature of reaction products that may lead to characterization of post-translational modification patterns, disulfide bond structures, and cross-linking in proteins or protein complexes. The most important unique feature of this program is the ability to perform multistage experiments in any user-defined order, thus allowing the researcher to vary experimental approaches that can be conducted in the laboratory. Several features of VIRTUALMSLAB are demonstrated by mapping both disulfide bonds and artificially introduced protein cross-links. It is shown that chemical cleavage at aspartate residues in the protease resistant RNase A, followed by tryptic digestion can be optimized so that the rigid protein breaks up into MALDI-MS detectable fragments, leaving the disulfide bonds intact. We also show the mapping of a number of chemically introduced cross-links in the NK1 domain of hepatocyte growth factor/scatter factor. The VIRTUALMSLAB program was used to explore the limitation and potential of mass spectrometry for cross-link studies of more complex biological assemblies, showing the value of high performance instruments such as a Fourier transform mass spectrometer. The program is freely available upon request.     

Caenorhabditis elegans: Characters of negatively charged groups on the cuticle and intestine

Partial characterization of carboxyl, sulfate, and phosphate groups on the Caenorhabditis elegans cuticle and intestinal microvilli was achieved by en face labeling of floating cryosections at two pH levels and specific blockage of sulfate groups by Alcian blue. All negatively charged groups on the cuticle and intestinal microvilli labeled heavily at pH 7.2–7.4. Pretreatment to block sulfate groups followed by ferritin labeling at pH 7.2–7.4 gave a 35% reduction of binding on the cuticle and an 80% reduction in binding on the microvilli. At pH 1.8 or 2.5, only the sulfate groups labeled as shown by the complete abolition of labeling on the cuticle and the microvilli following blockage of the sulfate groups. Molecules with accessible sulfate groups were distributed in clusters throughout the cortical layer of the cuticle, were present in the struts of the median layer but were absent from the basal layer. The advantages of applying molecular probes to cryosections as compared to sections prepared by standard electron microscopical techniques are discussed.     

Efficient in vivo gene delivery by the negatively charged complexes of cationic liposomes and plasmid DNA

We examined changes in zeta potential (the surface charge density, zeta) of the complexes of liposome (nmol)/DNA (microg) (L/D) formed in water at three different ratios (L/D=1, 10 and 20) by changing the ionic strength or pH to find an optimum formulation for in vivo gene delivery. At high DNA concentrations, zeta of the complexes formed in water at L/D=10 was significantly lowered by adding NaCl (zeta=+8.44+/-3.1 to -27.6+/-3.5 mV) or increasing pH from 5 (zeta=+15.3+/-1.0) to 9 (zeta=-22.5+/-2.5 mV). However, the positively charged complexes formed at L/D=20 (zeta=+6.2+/-3.5 mV) became negative as NaCl was added at alkaline pH as observed in medium (zeta=-19.7+/-9.9 mV). Thus, the complexes formed in water under the optimum condition were stable and largely negatively charged at L/D=1 (zeta=-58.1+/-3.9 mV), unstable and slightly positively charged at L/D=10 (zeta=+8.44+/-3.7 mV), and unstable and largely positively charged at L/D=20 (zeta=+24.3+/-3.6 mV). The negatively charged complexes efficiently delivered DNA into both solid and ascitic tumor cells. However, the positively charged complexes were very poor in delivering DNA into solid tumors, yet were efficient in delivering DNA into ascitic tumors grown in the peritoneum regardless of complex size. This slightly lower gene transfer efficiency of the negatively charged complexes can be as efficient as the positively charged ones when an injection is repeated (at least two injections), which is the most common case for therapy regimes. The results indicate that optimum in vivo lipofection may depend on the site of tumor growth.     

Cationic liposome and plasmid DNA complexes formed in serum-free medium under optimum transfection condition are negatively charged

In medium where in vitro transfection is routinely performed, DC-chol liposomes alone were nearly neutral, whereas the DC-chol liposome/DNA complexes were largely negatively charged which changed only slightly at all [liposome]/[DNA] ratios (zeta=-27.1 to -21.8 mV). Three other commercial transfection reagents, Lipofectin(R), LipofectAMINE 2000, and SuperFect, were also largely negatively charged when complexed with DNA. The aggregation of liposomes in medium was prevented by the addition of DNA. Incubation of the complexes in medium did not change their size, charge or lipofection activity for 30 min. These results suggest that, in medium, the liposome/DNA complexes were formed at the time of mixing with negative charges.     

Comparative studies on the effects of pH and Ca2+ on bilayers of various negatively charged phospholipids and their mixtures with phosphatidylcholine.

(1) The thermotropic behaviour of dimyristoyl phosphatidylglycerol, phosphatidylserine, phosphatidic acid and phosphatidylcholine was investigated by differential scanning calorimetry and freeze-fracture electron microscopy as a function of pH and of Ca2+ concentration. (2) From the thermotropic behaviour as a function of pH, profiles could be constructed from which apparent pK values of the charged groups of the lipids could be determined. (3) Excess Ca2+ induced a shift of the total phase transition in 14 : 0/14 : 0-glycerophosphocholine and 14 : 0/14 : 0-glycerophosphoglycerol mixtures. In 14 : 0/14 : 0-glycerophosphocholine bilayers containing 16 : 0/16 : 0-glycerophosphoglycerol lateral phase separation was induced by Ca2+. (4) Up to molar ratios of 1 : 2 of 14 : 0/14 : 0-glycerophosphoserine to 14 : 0/14: 0-glycerophosphocholine, excess Ca2+ induced lateral phase separation. Addition to mixtures of higher molar ratios caused segregation into different structures: the liposome organization and the stacked lamellae/cylindrical organization. (5) Addition of excess Ca2+ to mixtures of 14 : 0/14 : 0-glycerophosphocholine and 14 : 0/14 : 0-phosphatidic acid caused, independent of the molar ratio, separation into two structural different organizations. (6) The nature of Ca2+-induced changes in bilayers containing negatively charged phospholipids is strongly dependent on the character of the polar headgroup of the negatively charged phospholipid involved.     

Targeted liposomes in fungi: Modifying the therapeutic index of amphotericin b by its incorporation into negatively charged liposomes

Abstract

The mechanism of action of liposome—incorporated amphotericin B (ABLC) is not well understood. Most studies to date have dealt with the role of liposome size, lipid composition, and fluidity on ABLC toxicity and activity. However, little is known about the behavior of ABLC once injected into the circulation. This review describes the behavior of ABLC, both as a particle and in a lipophilic system, following intravenous administration in an attempt to better understand the mechanisms involved in ABLC's enhanced therapeutic index. Our data suggest that once ABLC is injected into the circulation, two major processes probably occur: (1) exchange with lipoproteins: both the liposomal lipid and the liposome—associated AmpB are actively exchanged with lipoproteins; and (2) phagocytosis: liposomes are taken up by phagocytes that potentially carry the internalized ABLC to infection sites. As a result of these interactions, ABLC is more effectively delivered to fungal cells where ABLC is released from the phagocyte and AmpB is liberated from the lipid by fungal lipases. Furthermore, ABLC predominantly distributes into high—density lipoproteins (HDL) following incubation in human serum for 1 hour at 37°C. This HDL—associated ABLC is less toxic to renal cells than either AmpB or LDL—associated AmpB because of the low level of expression of HDL receptors on renal cells. These findings demonstrate that the interaction of ABLC with plasma lipoproteins and blood phagocytes may be responsible for ABLC's enhanced therapeutic index.     

SecA insertion into phospholipids is stimulated by negatively charged lipids and inhibited by ATP: a monolayer study.

SecA-lipid interactions are believed to be important for the translocation of precursor proteins across the inner membrane of Escherichia coli [Lill, R., Dowhan, W., & Wickner, W. (1990) Cell 60, 271-280]. SecA insertion into the phospholipid bilayer could a role in this process. We investigated this possibility by studying the interactions between SecA and different phospholipids using the monolayer technique. It was established that SecA is surface-active and can insert into lipid monolayers. This insertion was greatly enhanced by the negatively charged lipids DOPG and Escherichia coli cardiolipin. Insertion of SecA into these negatively charged lipids could be detected up to initial surface pressures of 34 mN/m for DOPG and 36 mN/m for Escherichia coli cardiolipin, implying a possible role for negatively charged lipids in the insertion of SecA in biological membranes. High salt concentrations did not inhibit the SecA insertion into DOPG monolayers, suggesting not only an electrostatic but also a hydrophobic interaction of SecA with the lipid monolayer. ATP decreased both the insertion (factor 2) and binding (factor 3) of SecA to DOPG monolayers. ADP and phosphate gave a decrease in the SecA insertion to the same extent as ATP, but the binding of SecA was only slightly reduced. AMP-PNP and ATP-gamma-S did not have large effects on the insertion or on the binding of SecA to DOPG monolayers. The physiological significance of these results in protein translocation is discussed.     

Enzyme therapy VI: Comparative in vivo fates and effects on lysosomal integrity of enzyme entrapped in negatively and positively charged liposomes

Entrapment of enzyme in liposomes, biodegradable lipid vesicles, offers an intriguing strategy for the intracellular delivery of these macromolecules to the lysosomal apparatus for enzyme replacement endeavors in selected lysosomal storage diseases. Therefore, the in vivo tissue and subcellular fate and effect on the subcellular distribution of endogenous lysosomal hydrolases was determined following intravenous administration of β-glucuronidase entrapped in positively and negatively charged liposomes into C3H/HeJ β-glucuronidase-deficient mice. Enzyme entrapped in negatively charged liposomes was rapidly cleared from the circulation ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 4}\text{min}$); maximal tissue recovery, 75% of dose, was detected in the liver at 1 h, was maintained for 48 h and then gradually declined to non-detectable levels by 8 days. A similar circulatory clearance and reciprocal hepatic uptake was observed for positively charged liposomes; however, the β-glucuronidase was retained in murine liver for 11 days. Significant activity, 15% of dose, was found in the kidneys up to 1 and 4 days post-injection of positively and negatively charged liposomes, respectively. No activity was recovered in neural or other visceral tissues except in spleen and lungs (?5% of dose). Exogenous β-glucuronidase activity administered in negatively charged liposomes was primarily localized in the lysosomally-enriched hepatic subcellular fraction, compared to the predominantly soluble localization of exogenous activity entrapped in positively charged liposomes. Administration of negatively charged liposomes caused no detectable change in the subcellular localization of several endogenous lysosomal hydrolase activities compared to their distribution in untreated mice. In contrast, a marked but temporary translocation of these hydrolase activities into the soluble fraction was observed following the administration of positively charged liposomes, identifying possible deleterious effects on cellular physiology.     

Thermotropic properties of mixtures of negatively charged phospholipids with cholesterol in the presence and absence of Li+ or Ca2+ ions

Mixtures of cholesterol with dipalmitoylphosphatidylserine or phosphatidic acid were investigated by differential scanning calorimetry. As in mixtures of natural phosphatidylserine with cholesterol (Bach, D. (1984) Chem. Phys. Lipids 35, 385-392), also here phase separation of cholesterol at molar ratios of 2:1 (phospholipid:cholesterol) and below is observed. The limited solubility of cholesterol in negatively charged phospholipids is found to be independent of the nature of the acyl chain residues, and independent of whether the negative charge resides on both COO- and PO- groups (as in phosphatidylserine) or on PO- only (as in phosphatidic acid). The separate cholesterol phase is also seen by DSC in mixtures of natural phosphatidylserine or phosphatidic acid with cholesterol in the presence of Ca2+; and in phosphatidylserine/cholesterol mixtures in the presence of Li+, by DSC and X-ray diffraction.     

Role of negatively charged amino acids in beta 4 F-loop in activation and desensitization of alpha 3 beta 4 rat neuronal nicotinic receptors

The role of negatively charged amino acids in the F-loop of the beta 4 subunit in channel activation and desensitization was studied using the patch-clamp technique. The selected amino acids were changed to their neutral analogs via point mutations. Whole-cell currents were recorded in COS cells transiently transfected with the alpha 3 beta 4 nicotinic acetylcholine receptor. The application of acetylcholine (ACh), nicotine (Nic), cytisine (Cyt), carbamylcholine (CCh) and epibatidine (Epi) to cells clamped at -40 mV produced inward currents which displayed biphasic desensitization. The EC50 of Epi and Nic were increased by a factor of 3-6 due to mutations D191N or D192N. Only Epi remained an agonist in the double-mutated receptors with EC50 increased 17-fold. The interaction of the receptors with the competitive antagonist (+)tubocurarine (TC) was weakened almost 3-fold in the double-mutated receptors. The mutations increased the proportion of the slower desensitization component and increased the response plateau, resulting in decreased receptor desensitization. The double mutation substantially accelerated the return from long-term desensitization induced by Epi.     

The structures of L-rhamnose isomerase from Pseudomonas stutzeri in complexes with L-rhamnose and D-allose provide insights into broad substrate specificity

Pseudomonas stutzeril-rhamnose isomerase (P. stutzeri L-RhI) can efficiently catalyze the isomerization between various aldoses and ketoses, showing a broad substrate specificity compared to L-RhI from Escherichia coli (E. coli L-RhI). To understand the relationship between structure and substrate specificity, the crystal structures of P. stutzeri L-RhI alone and in complexes with l-rhamnose and d-allose which has different configurations of C4 and C5 from l-rhamnose, were determined at a resolution of 2.0 Å, 1.97 Å, and 1.97 Å, respectively. P. stutzeri L-RhI has a large domain with a (β/α)₈ barrel fold and an additional small domain composed of seven α-helices, forming a homo tetramer, as found in E. coli L-RhI and d-xylose isomerases (D-XIs) from various microorganisms. The β1-α1 loop (Gly60-Arg76) of P. stutzeri L-RhI is involved in the substrate binding of a neighbouring molecule, as found in D-XIs, while in E. coli L-RhI, the corresponding β1-α1 loop is extended (Asp52-Arg78) and covers the substrate-binding site of the same molecule. The complex structures of P. stutzeri L-RhI with l-rhamnose and d-allose show that both substrates are nicely fitted to the substrate -binding site. The part of the substrate-binding site interacting with the substrate at the 1, 2, and 3 positions is equivalent to E. coli L-RhI, and the other part interacting with the 4, 5, and 6 positions is similar to D-XI. In E. coli L-RhI, the β1-α1 loop creates an unique hydrophobic pocket at the the 4, 5, and 6 positions, leading to the strictly recognition of l-rhamnose as the most suitable substrate, while in P. stutzeri L-RhI, there is no corresponding hydrophobic pocket where Phe66 from a neighbouring molecule merely forms hydrophobic interactions with the substrate, leading to the loose substrate recognition at the 4, 5, and 6 positions.     

Ion-pair mechanism in square planar substitution. Reactivity of cationic platinum (II) complexes with negatively charged nucleophiles in solvents of high, medium and low polarity

The rates of displacement of dimethyl sulfoxide from the cation [Pt(phen) (CH₃) (Me₂SO)]⁺ by a series of uncharged and negatively charged nucleophiles have been measured in a methanol/water (19:1 vol./vol.) mixture. The starting complex and the reaction products were characterized either as solids or in solution by their IR and ¹H NMR spectra. The substitution reactions take place by way of a direct bimolecular attack of the ligand on the substrate. The sequence of reactivity observed is as expected on the basis of a nucleophilicity scale relevant for + 1 charged substrates ([Pt(en) (NH₃)Cl]⁺ used as standard). The difference of reactivity between the first (t-BuNH₂) and the last (SeCN⁻) members of the series spans five orders of magnitude. The value measured for the nucleophilic discrimination (1.55) is the highest found so far for cationic substrates. This is a result of the easy transfer of some of the electron density brought in by the incoming ligand into the ancillary ligands. When the reaction is carried out in a series of protic and dipolar aprotic solvents, using chloride ion as nucleophile, the rate of formation of [Pt (phen) (CH₃)Cl] is dominated by the extent of solvation of Cl⁻, as measured by its values of the Gibbs molar energy of transfer Δ_tG⁰. Conductivity measurements at 25°C in dichloromethane were fitted to the Fuoss equation and the values of the dissociation constants K_d for the ion pairs were calculated as follows: 2.27 × 10⁻⁵ M for Bu₄NCl, 2.75 × 10⁻⁵ M for Bu₄NSCN and 17.05 × 10⁻⁵ M for [Pt(phen) (CH₃) (Me₂SO)]PF₆. The pseudo-first-order rate constants k_obs for the reactions with Bu₄NCl, Bu₄NBr, Bu₄NSCN and Bu₄NI showed a curvilinear dependence on the concentration of the salt which levels off very soon (at concentrations higher than 0.005 M the kinetics are zero order in [Bu₄NX]). On addition of the inert electrolyte Bu₄NPF₆ the rates slow down and the kinetics follow the rate law k_obs = kK_ip[Bu₄NX]/[Bu₄NPF₆] + K_ip[Bu₄NX]). These findings fit well with a reaction scheme which involves a pre-equilibrium K_ip between ion pairs, followed by unimolecular substitution within the contact ion pair [Pt(phen) (CH₃) (Me₂SO)X]_ip. Values of the equilibrium constants K_ip for ion-pair exchange and of the internal substitution rates k were derived. The latter showed that the discrimination in reactivity between Cl⁻, Br⁻, SCN⁻ and I⁻ is greatly reduced with respect to aqueous solutions. The reason behind this may be desolvation of the ions coupled to the fact that a contact ion pair is already at a certain distance along the reaction coordinate in the direction of the transition state. Applications of the special salt effect and of ion pairing to synthesis are discussed.     

Introduction of additional thiol groups into glucoamylase in Aspergillus Awamori and their effect on the thermal stability and catalytic activity of the enzyme

Five mutant forms of glucoamylase (GA) from the filamentous fungus Aspergillus awamori with artificial disulfide bonds (4D-G137A\A14C, 6D-A14C\Y419C\G137A, 10D-V13C\G396C, 11D-V13C\G396C\A14C\Y419C\G137A, and 20D-G137A\A246C\A14C) were constructed using molecular modeling simulations and experimentally tested for thermostability. The introduction of two additional disulfide bonds between its first and thirteenth α-helices and that of the loop located close to a catalytic residue—E400—made it possible to assess the effects of disulfide bridges on protein thermostability. The mutant proteins with combined amino acid substitutions G137A\A14C, V13C\G396C\A14C\Y419C\G137A, and G137A\A246C\A14C showed higher thermal stability as compared to the wild-type protein. At the same time, new disulfide bridges in the mutant A14C\Y419C\G137A and V13C\G396C proteins led to the destabilization of their structure and the loss of thermal stability.     

Expression profiles of growth-related genes in two Nile tilapia strains and their crossbred provide insights into introgressive breeding effects

The Nile tilapia (Oreochromis niloticus) is a prominent farmed fish in aquaculture worldwide. Crossbreeding has recently been carried out between the Red-Stirling and the wt Chitralada strains of Nile tilapia, producing a heterotic hybrid (7/8 Chitralada and 1/8 Red-Stirling) that combines the superior growth performance of the Chitralada with the reddish coloration of the Red-Stirling strain. While classical selective breeding and crossbreeding strategies are well known, the molecular mechanisms underlying the phenotypic expression of economically advantageous traits in tilapia remain largely unknown. Molecular investigations have shown that variable expression of growth hormone (gh), insulin-like growth factors (igf1 and 2) and somatolactin (smtla) – components of the growth hormone/insulin-like growth factor (GH/IGF) axis – and myostatin (mstn) genes can affect traits of economic relevance in farmed animals. The aim of this study was to assess and compare the gene expression signature among Chitralada, Red-Stirling and their backcross hybrid in order to gain insights into the effects of introgressive breeding in modulation of the GH/IGF axis. Gene expression analyses in distinct tissues showed that most genes of the GH/IGF axis were up-regulated and mstn was down-regulated in backcross animals in comparison with Red-Stirling and Chitralada animals. These gene expression profiles revealed that backcross animals displayed a distinctive expression signature, which attests to the effectiveness of the introgressive breeding technique. Our findings also suggest that the GH/IGF axis and mstn genes might be candidate markers for fish performance and prove useful within genetic improvement programs aimed at the production of superior-quality tilapia strains using introgressive breeding.     

Expression and functional characterization of mutant human CXCR4 in insect cells: role of cysteinyl and negatively charged residues in ligand binding

Human CXCR4 was expressed in Sf9 insect cells using the Bac-to-Bac baculovirus expression system. The recombinant receptor exhibited ligand binding activities with a K(d) value (3.3 nM) comparable to that of the native receptor. The role of four conserved cysteinyl residues was explored by site-directed mutagenesis. Each cysteine was individually changed to an alanine residue. All of the four mutants showed decreased ligand binding activity with increased K(d) values although comparable levels of receptor expression were observed. These results suggest that each of these four cysteinyl residues may be important for the ligand binding of the receptor. Evidence suggests that the ionic interaction may be involved in ligand binding. Point mutation of several relatively conserved acidic residues (Asp-10, Asp-262, Glu-275, and Glu-277) to an alanine residue greatly decreased the ligand binding activity and affinity. Since SDF-1alpha is a highly basic protein, these acidic residues may interact with the basic residues of SDF-1alpha by ionic pairing in addition to other molecular interactions and play an important role in ligand binding.     

Highly efficient "tight fit" immobilization of alpha-chymotrypsin in mesoporous MCM-41: a novel approach using precursor immobilization and activation

The zymogen alpha-chymotrypsinogen A is bound to mesoporous silica MCM-41 with a protein loading of 170 mg/g solid (MCM-Z) by a simple stirring in aqueous tris-HCl buffer (pH 7.2). The bound zymogen is then activated with trypsin to obtain alpha-chymotrypsin immobilized on MCM-41 (MCM-E.I) that displays an effective enzyme activity corresponding to 65 mg protein/g of solid support (3250 BTEE units/g). A direct immobilization of commercially available alpha-chymotrypsin (MCM-E.II) gives lower loading (1250 BTEE units/g). Protein content of the solid support after immobilization is confirmed by thermogravimetric analysis (TGA). The enzyme is tightly bound to the support and can be used over 100 recycles over 1 week in aqueous as well as reverse micellar media. The immobilized enzyme (MCM-E.I) has been used for resolution of N-acetyl-dl-amino acid esters and racemic trans-4-methoxy-3-phenylglycidic acid (PGA) methyl ester.