Photopolymerized phospholipid vesicles Stability and retention of hydrophilic and hydrophobic marker substances

Multilamellar vesicles were formed from photopolymerizable analogs of phosphatidylcholine. The polymerized vesicles differed markedly from conventional vesicles in terms of their stability to mechanical and chemical perturbations. Thus, polymerized vesicles, but not conventional ones, retained their overall physical integrity subsequent to ultrasonication or to exposure to organic solvents or detergents. Treatment of photopolymerized vesicles with detergents such as sodium dodecyl sulfate caused the release of entrapped hydrophilic solutes; however, lipophilic solutes were retained by the polymerized vesicles under circumstances in which conventional vesicles were completely solubilized. Thus, photopolymerized phospholipid membranes represent hybrid entities which seem to blend some of the characteristics of conventional lipid bilayers with properties more typical of polymer membranes.     

Bioaffinity-based an inexpensive and high yield procedure for the immobilization of turnip (Brassica rapa) peroxidase

This study demonstrates the immobilization of carbohydrate containing turnip peroxidase on an inexpensive bioaffinity adsorbent, Concanavalin A-cellulose support. The bioaffinity support was prepared simply by incubating cellulose powder with jack bean extract at 4 degrees C. Cellulose powder adsorbed 30 mg concanavalin A/g of the matrix. Concanavalin A adsorbed cellulose has been employed for the simultaneous purification and immobilization of glycoenzymes directly from ammonium sulphate fractionated proteins of turnip. The obtained bioaffinity support was quite effective in high yield immobilization of peroxidase from turnip and it retained 672 U/g. Turnip peroxidase immobilized on concanavalin A-cellulose support retained 80% of the initial activity. Immobilized turnip peroxidase preparation was quite resistant against the denaturation mediated by pH, heat, urea, guanidinium-HCl, Surf Excel, cetyltrimethylammonium bromide and water-miscible organic solvents; dimethyl formamide, dioxane and n-propanol. Low concentration of detergents like Surf Excel and cetyltrimethylammonium bromide enhanced the activity of soluble and immobilized turnip peroxidase.     

Continuous production of NADP by immobilized Brevibacterium ammoniagenes cells.

Whole cells of Brevibacterium ammoniagenes IAM 1645 having the polyphosphate NAD-kinase were successfully immobilized in a polyacrylamide gel lattice. The immobilized cells were activated by treatment with organic solvents or detergents. The pH optimum of the immobilized cells for the production of NADP was 7.0, and divalent metal ions were required to maintain the elevated activity of polyphosphate NAD-kinase. Highly pure NADP was continuously produced in high yield by the immobilized cell column. The half-life of this column was about eight days.     

A gas chromatography–mass spectrometry method to monitor detergents removal from a membrane protein sample

In membrane protein biochemical and structural studies, detergents are used to mimic membrane environment and maintain functional, stable conformation of membrane proteins in the absence of lipid bilayers. However, detergent concentration, esp. molar ratio of membrane protein to detergent is usually unknown. Here, a gas chromatography–mass spectrometry selected ion monitoring (GC–MS-SIM) method was developed to quantify four detergents which are frequently used in membrane protein structural studies. To remove excessive detergents, a filtered centrifugation using Centricon tubes was applied. A membrane protein Ig-Beta fragment in four different detergent micelles was exemplified. Detergent concentrations in the upper and lower fraction of the Centricon tube were measured after each round of centrifugation. The results were very consistent to basic properties of detergent micelles in aqueous solvents. Therefore, coupling of GC–MS-SIM and detergent removal by Centricon tubes, detergents concentration, esp. molar ratio of membrane protein to detergent could be controlled, which will expedite membrane protein structural and biochemical studies.     

Production and immobilization of a novel thermoalkalophilic extracellular amylase from bacilli isolate

A Thermoalkalophilic amylase was produced from an environmental bacterial isolate. The enzyme was then immobilized through its amino groups onto the epoxy rings of magnetic poly glycidyl methacrylate [m-poly (GMA)] beads. The free enzyme was active within a large pH range, between 7 and 12 and displayed the optimum activity at 95°C and pH 10. The immobilization appeared to increase the stability of the enzyme as its bound form showed optimum activity at 105°C and pH 11.0. Kinetic studies demonstrated that immobilized enzyme had higher K(m) and lower V(max) values. The activity of the free and bound enzyme was determined, at 37°C and pH 10.0 and pH 11.0, respectively, in the presence of various organic solvents and detergents (5%, v/v). Results obtained indicated that detergents, sodium dodecyl sulfate (SDS) and TritonX-100, caused six fold increase and that various organic solvents also increased the activity of the amylase.     

Evaluation of deep eutectic solvents as new media for Candida antarctica B lipase catalyzed reactions

This study aimed at analyzing the advantages and limitations of several deep eutectic solvents (DESs) as ‘green solvents’ for biotransformation using immobilized Candida antarctica lipase B as catalyst. The transesterification of vinyl laurate was chosen as model reaction and the influence of substrate polarity was assessed using alcohols of various chain lengths. Results showed that grinding of immobilized lipase was essential parameters for good lipase activity. Moreover, in our model reaction some hydrogen-bond donor component from the DES can compete with the alcoholysis reaction. Indeed, side reactions were observed with DES based on dicarboxylic acid or ethylene glycol, leading to some limitations of their use. However, the results showed that other DESs such as choline chloride:urea and choline chloride:glycerol could exhibit high activity and selectivity making them promising solvents for lipase-catalyzed reactions. Finally, the best DES's specific activity – and stability up to five days incubation time – were analyzed and compared with conventional organic solvents. Experiments revealed that iCALB is less influenced by the chain length of alcohol in DES than organic solvents and it is preserves its activity with minimally destructive to protein structure.     

Bcl-xL forms two distinct homodimers at non-ionic detergents: implications in the dimerization of Bcl-2 family proteins

As the key regulator of apoptosis, Bcl-2 family protein controls the cell death by forming homo- or heterodimers among anti-apoptotic and pro-apoptotic members of this family. Here we have studied Bcl-x(L) homodimerization at different pH in the presence of various detergents and organic solvents. We found that both acidic and basic pHs are beneficial for Bcl-x(L) dimerization. High concentrations of non-ionic detergents and some organic solvents can significantly promote this event. In addition to non-covalently linked acidic-dimer as that formed at acidic pH, Bcl-x(L) formed disulphide-bonded detergent-dimer at neutral and basic pH when incubated with high concentrations of non-ionic detergents. The acidic-dimer retains the BH3 peptide binding activity, whereas the detergent-dimer does not. The formation of acidic-dimer and detergent-dimer implies that Bcl-x(L) may dimerize via two different pathways under certain conditions. The implications of these findings has been discussed with previous experimental results, which provides some new insight into the events and would help the experiment design and data interpretation when non-ionic detergents are used to study the dimerization and pore formation of Bcl-2 family proteins.     

Polyclonal antibodies mediated immobilization of a peroxidase from ammonium sulphate fractionated bitter gourd (Momordica charantia) proteins

Polyclonal antibody bound Sepharose 4B support has been exploited for the immobilization of bitter gourd peroxidase directly from ammonium sulphate precipitated proteins. Immunoaffinity immobilized bitter gourd peroxidase exhibited high yield of immobilization. IgG-Sepharose 4B bound bitter gourd peroxidase showed a higher stability against heat, chaotropic agents (urea and guanidinium chloride), detergents (cetyl trimethyl ammonium bromide and Surf Excel), proteolytic enzyme (trypsin) and water-miscible organic solvents (propanol, THF and dioxane). The activity of immobilized bitter gourd peroxidase was significantly enhanced in the presence of cetyl trimethyl ammonium bromide and after treatment with trypsin as compared to soluble enzyme.     

Nanoscale analysis of supported lipid bilayers using atomic force microscopy

During the past 15 years, atomic force microscopy (AFM) has opened new opportunities for imaging supported lipid bilayers (SLBs) on the nanoscale. AFM offers a means to visualize the nanoscale structure of SLBs in physiological conditions. A unique feature of AFM is its ability to monitor dynamic events, like bilayer alteration, remodelling or digestion, upon incubation with various external agents such as drugs, detergents, proteins, peptides, nanoparticles, and solvents. Here, we survey recent progress made in the area.     

Enhanced reactivity of Rhizopus oryzae lipase displayed on yeast cell surfaces in organic solvents: potential as a whole-cell biocatalyst in organic solvents

Immobilization of enzymes on some solid supports has been used to stabilize enzymes in organic solvents. In this study, we evaluated applications of genetically immobilized Rhizopus oryzae lipase displayed on the cell surface of Saccharomyces cerevisiae in organic solvents and measured the catalytic activity of the displayed enzyme as a fusion protein with alpha-agglutinin. Compared to the activity of a commercial preparation of this lipase, the activity of the new preparation was 4.4 x 10(4)-fold higher in a hydrolysis reaction using p-nitrophenyl palmitate and 3.8 x 10(4)-fold higher in an esterification reaction with palmitic acid and n-pentanol (0.2% H2O). Increased enzyme activity may occur because the lipase displayed on the yeast cell surface is stabilized by the cell wall. We used a combination of error-prone PCR and cell surface display to increase lipase activity. Of 7,000 colonies in a library of mutated lipases, 13 formed a clear halo on plates containing 0.2% methyl palmitate. In organic solvents, the catalytic activity of 5/13 mutants was three- to sixfold higher than that of the original construct. Thus, yeast cells displaying the lipase can be used in organic solvents, and the lipase activity may be increased by a combination of protein engineering and display techniques. Thus, this immobilized lipase, which is more easily prepared and has higher activity than commercially available free and immobilized lipases, may be a practical alternative for the production of esters derived from fatty acids.     

In vitro expression and biochemical characterization of juvenile hormone esterase from Manduca sexta

Juvenile hormone esterase (JHE) is a selective enzyme that hydrolyzes the methyl ester of juvenile hormone. This enzyme plays an important role in the regulation of metamorphosis in caterpillars, and is implicated in additional roles in development and reproduction in this and other orders of insect. The full length coding region of the JHE cDNA from Manduca sexta was subcloned into the baculovirus AcMNPV genome under the control of the p10 promoter. The recombinant virus demonstrated the expression of high levels of JHE activity when infected into Hi5 cells from Trichoplusia ni. The recombinant protein was partially purified by anion exchange chromatography and its biochemical characterization showed similar features to the wild type protein. The recombinant JHE has an estimated MW of 66500 Da. Some heterogeneity with the enzyme was observed when analyzed by isoelectric focusing, although the peak of JHE activity was observed at pI=6.0. It is highly sensitive to trifluoroketone inhibitors and certain phosphoramidothiolates, while relatively insensitive to other common esterase inhibitors. Incubating the enzyme with various organic solvents and detergents showed that the enzyme is activated at lower concentrations of solvents/detergents and remains significantly active even at high concentrations. The high tolerance of organic solvents may make this JHE enzyme useful in future applications as a synthetic catalyst.     

Effects of Lipid-Analog Detergent Solubilization on the Functionality and Lipidic Cubic Phase Mobility of the Torpedo californica Nicotinic Acetylcholine Receptor

Over the past three decades, the Torpedo californica nicotinic acetylcholine receptor (nAChR) has been one of the most extensively studied membrane protein systems. However, the effects of detergent solubilization on nAChR stability and function are poorly understood. The use of lipid-analog detergents for nAChR solubilization has been shown to preserve receptor stability and functionality. The present study used lipid-analog detergents from phospholipid-analog and cholesterol-analog detergent families for solubilization and affinity purification of the receptor and probed nAChR ion channel function using planar lipid bilayers (PLBs) and stability using analytical size exclusion chromatography (A-SEC) in the detergent-solubilized state. We also examined receptor mobility on the lipidic cubic phase (LCP) by measuring the nAChR mobile fraction and diffusion coefficient through fluorescence recovery after photobleaching (FRAP) experiments using lipid-analog and non-lipid-analog detergents. Our results show that it is possible to isolate stable and functional nAChRs using lipid-analog detergents, with characteristic ion channel currents in PLBs and minimal aggregation as observed in A-SEC. Furthermore, fractional mobility and diffusion coefficient values observed in FRAP experiments were similar to the values observed for these parameters in the recently LCP-crystallized β(2)-adrenergic receptor. The overall results show that phospholipid-analog detergents with 16 carbon acyl-chains support nAChR stability, functionality and LCP mobility.     

Detergents for the stabilization and crystallization of membrane proteins

The use of detergents for the structural study of membrane proteins is discussed with an emphasis on practical issues relating to membrane solubilization, protein aggregation, detergent purity and detergent quantitation. Detergents are useful reagents as mimics of lipid bilayers because of their self-assembling properties, but as a result, they have complex properties in solution. It can be difficult to maintain a solubilized membrane protein in a native conformational state, and the non-specific aggregation of detergent-solubilized proteins is a common problem. Empirical "stability screens" can be helpful in choosing which detergents, and which detergent concentrations, may be optimal for a given system.     

A bifunctional monolithic column for combined protein preconcentration and digestion for high throughput proteomics research

Enzymatic digestion of proteins is a key step in protein identification by mass spectrometry (MS). Traditional solution-based protein digestion methods require long incubation times and are limitations for high throughput proteomics research. Recently, solid phase digestion (e.g. trypsin immobilization on solid supports) has become a useful strategy to accelerate the speed of protein digestion and eliminate autodigestion by immobilizing and isolating the enzyme moieties on solid supports. Monolithic media is an attractive support for immobilization of enzymes due to its unique properties that include fast mass transfer, stability in most solvents, and versatility of functional groups on the surfaces of monoliths. We prepared immobilized trypsin monolithic capillaries for on-column protein digestion, analyzed the digested peptides through LC/FTICR tandem MS, and compared peptide mass fingerprinting by MALDI-TOF-MS. To further improve the digestion efficiency for low abundance proteins, we introduced C4 functional groups onto the monolith surfaces to combine on-column protein enrichment and digestion. Compared with immobilized trypsin monolithic capillaries without C4, the immobilized trypsin-C4 monolith showed improved digestion efficiency. A mechanism for increased efficiency from the combination of sample enrichment and on-column digestion is also proposed in this paper. Moreover, we investigated the effects of organic solvent on digestion and detection by comparing the observed digested peptide sequences. Our data demonstrated that all columns showed good tolerance to organic solvents and maintained reproducible enzymatic activity for at least 30 days.     

Regeneration of ethyl parathion antibodies for repeated use in immunosensor: a study on dissociation of antigens from antibodies

Reliable analysis using an immunosensor strongly depends on the specificity, activity, and sensitivity of the antibody. Immobilization of antibody on the solid matrix enables its repeated use, for which it is required to dissociate the antigens and antigen-enzyme conjugate from the immobilized antibody matrix after each use and while doing so, a maximum retention of activity and specificity are crucial requirements. In the present investigation, on the development of an immunosensor for the organophosphorus pesticide ethyl parathion (EP) using EP antibodies, different dissociating agents such as organic solvents, detergents and acidic buffers, that is, dimethyl sulphoxide (DMSO), Tween-20, cetyl trimethylammonium bromide (CTAB), methanol, chloroform, guanidium chloride (GdmCl), glycine-HCl (Gly-HCl) buffer in the pH range of 1.5-3.0, pierce buffer and combination of DMSO and methanol in phosphate buffer and Gly-HCl buffer and salts like NaCl and MgCl2 were used. Generally about 50-60% dissociation was obtained with some degree of denaturation of the antibody immobilized on the sepharose matrix. However, 1% DMSO in combination with 0.2 M Gly-HCl buffer at a pH of 2.3 showed 97% dissociation and the immobilized antibody retained sufficient activity to carry out 14 reproducible assays for EP.     

Stability of the larvicidal activity of Bacillus thuringiensis subsp. israelensis: amino acid modification and denaturants

The Bacillus thuringiensis subsp. israelensis mosquito larvicidal toxin is not a sulfhydryl-activated toxin. The protein disulfide bonds were cleaved and blocked without loss of toxicity. In contrast, modification of the lysine side chains eliminated toxicity. Additionally, the toxin was resistant to high concentrations of salt (8 M NaBr), organic solvents (40% methanol), denaturants (4 M urea), and neutral detergents (10% Triton X-100). However, it was inactivated by both positively and negatively charged detergents and by guanidine hydrochloride.     

Stability of the larvicidal activity of Bacillus thuringiensis subsp. israelensis: amino acid modification and denaturants.

The Bacillus thuringiensis subsp. israelensis mosquito larvicidal toxin is not a sulfhydryl-activated toxin. The protein disulfide bonds were cleaved and blocked without loss of toxicity. In contrast, modification of the lysine side chains eliminated toxicity. Additionally, the toxin was resistant to high concentrations of salt (8 M NaBr), organic solvents (40% methanol), denaturants (4 M urea), and neutral detergents (10% Triton X-100). However, it was inactivated by both positively and negatively charged detergents and by guanidine hydrochloride.     

Analysis of a G protein-coupled receptor for neurotensin by liquid chromatography-electrospray ionization-mass spectrometry

The type 1 neurotensin receptor (NTS1) belongs to the G protein-coupled receptor (GPCR) family. GPCRs are involved in important physiological processes, but for many GPCRs ligand binding sites and other structural features have yet to be elucidated. Comprehensive analyses by mass spectrometry (MS) could address such issues, but they are complicated by the hydrophobic nature of the receptors. Recombinant NTS1 must be purified in the presence of detergents to maintain solubility and functionality of the receptor, to allow testing of ligand, or to allow G protein interaction. However, detergents are detrimental to MS analyses. Hence, steps need to be taken to substitute the detergents with MS-compatible polar/organic solvents. Here we report the characterization of NTS1 by electrospray ionization (ESI)-MS with emphasis on methods to transfer intact NTS1 or its proteolytic peptides into compatible solvents by protein precipitation and liquid chromatography (LC) prior to ESI-MS analyses. Molecular mass measurement of intact recombinant NTS1 was performed using a mixture of chloroform/methanol/aqueous trifluoroacetic acid as the mobile phase for size exclusion chromatography-ESI-MS analysis. In a separate experiment, NTS1 was digested with a combination of cyanogen bromide and trypsin and/or chymotrypsin. Subsequent reversed phase LC-ESI-tandem MS analysis resulted in greater than 80% sequence coverage of the NTS1 protein, including all seven transmembrane domains. This work represents the first comprehensive analysis of recombinant NTS1 using MS.     

Enhanced Reactivity of Rhizopus oryzae Lipase Displayed on Yeast Cell Surfaces in Organic Solvents: Potential as a Whole-Cell Biocatalyst in Organic Solvents

Immobilization of enzymes on some solid supports has been used to stabilize enzymes in organic solvents. In this study, we evaluated applications of genetically immobilized Rhizopus oryzae lipase displayed on the cell surface of Saccharomyces cerevisiae in organic solvents and measured the catalytic activity of the displayed enzyme as a fusion protein with α-agglutinin. Compared to the activity of a commercial preparation of this lipase, the activity of the new preparation was 4.4 × 10⁴-fold higher in a hydrolysis reaction using p-nitrophenyl palmitate and 3.8 × 10⁴-fold higher in an esterification reaction with palmitic acid and n-pentanol (0.2% H₂O). Increased enzyme activity may occur because the lipase displayed on the yeast cell surface is stabilized by the cell wall. We used a combination of error-prone PCR and cell surface display to increase lipase activity. Of 7,000 colonies in a library of mutated lipases, 13 formed a clear halo on plates containing 0.2% methyl palmitate. In organic solvents, the catalytic activity of 5/13 mutants was three- to sixfold higher than that of the original construct. Thus, yeast cells displaying the lipase can be used in organic solvents, and the lipase activity may be increased by a combination of protein engineering and display techniques. Thus, this immobilized lipase, which is more easily prepared and has higher activity than commercially available free and immobilized lipases, may be a practical alternative for the production of esters derived from fatty acids.