GroESL overexpression imparts Escherichia coli tolerance to i-, n-, and 2-butanol, 1,2,4-butanetriol and ethanol with complex and unpredictable patterns

Strain tolerance to toxic metabolites remains a limiting issue in the production of chemicals and biofuels using biological processes. Here we examined the impact of overexpressing the autologous GroESL chaperone system with its natural promoter on the tolerance of Escherichia coli to several toxic alcohols. Strain tolerance was examined using both a growth assay as well as viable cell counts employing a CFU (colony-forming unit) assay. GroESL over expression enhanced cell growth to all alcohols tested, including a 12-fold increase in total growth in 48-h cultures under 4% (v/v) ethanol, a 2.8-fold increase under 0.75% (v/v) n-butanol, a 3-fold increase under 1.25% (v/v) 2-butanol, and a 4-fold increase under 20% (v/v) 1,2,4-butanetriol. GroESL overexpression resulted in a 9-fold increase in CFU numbers compared to a plasmid control strain after 24 h of culture under 6% (v/v) ethanol, and a 3.5-fold and 9-fold increase for culture under 1% (v/v) n-butanol and i-butanol, respectively. The toxicity of the alcohols was examined against their octanol–water partition coefficient, a measure commonly used to predict solvent toxicity. For both the control and the GroESL overexpressing strains, the calculated membrane concentration of each alcohol based on the octanol–water partition coefficient could be correlated, but with different patterns, to the impact of the various alcohols on cell growth, but not on cell viability (CFUs). Our data suggest a complex pattern of growth inhibition and differential protection by GroESL overexpression depending on the specific alcohol molecule. Overall, however, GroESL overexpression appears to provide molecule-agnostic tolerance to toxic chemicals.     

Improvement of hydrogenase enzyme activity by water-miscible organic solvents

Both stability and catalytic activity of the HynSL Thiocapsa roseopersicina hydrogenase in the presence of different water-miscible organic solvents were investigated. For all organic solvents under study the substantial raise in hydrogenase catalytic activity was observed. The stimulating effect of acetone and acetonitrile on the reaction rate rose with the increase in solvent concentration up to 80%. At certain concentrations of acetonitrile and acetone (60–80%, v/v in buffer solution) the enzyme activity was improved even 4–5 times compared to pure aqueous buffer. Other solvents (aliphatic alcohols, dimethylsulfoxide and tetrahydrofuran) improved the enzyme activity at low concentrations and caused enzyme inactivation at intermediate concentrations. The long-term incubation of the hydrogenase with aliphatic alcohols, dimethylsulfoxide and tetrahydrofuran at intermediate concentrations of the latter caused enzyme inactivation. The reduced form of hydrogenase was found to be much more sensitive to action of these organic solvents than the enzyme being in oxidized state. The hydrogenase is rather stable at high concentrations of acetone or acetonitrile during long-term storage: its residual activity after incubation in these solvents upon air within 30 days was about 50%, and immobilized enzyme remained at the 100% of its activity during this period.     

Interaction between glycophorin and ganglioside GM1 on liposomal membranes. Effect of the interaction on the susceptibility of membranes to HVJ

Liposomes could bind and fuse efficiently to human erythrocytes in the presence of HVJ when they contained glycophorin isolated from human erythrocytes (Umeda, M., et al. (1983) J. Biochem. 94, 1955). In the present work we demonstrated that HVJ-induced fusion between liposomes containing glycophorin and erythrocytes was suppressed when GM1 coexisted with glycophorin in the same liposomal membranes. Asialo-GM1 and other gangliosides such as GM3 and sialosylparagloboside did not affect the fusion between the liposomes and erythrocytes. An intermolecular interaction between glycophorin and GM1 was suggested by the ESR spectrum obtained from liposomes containing glycophorin and a ganglioside GM1 analog carrying a nitroxyl spin label in the fatty acyl chains (5SL-gangliosidoide). The overall splitting value (2A parallel) observed in the ESR spectrum of liposomes containing 5SL-gangliosidoide increased with increase of the amount of glycophorin, whereas 2A parallel of spin-labeled phosphatidylcholine was not changed. The increase of 2A parallel of 5SL-gangliosidoide suggests that the mobility of the fatty acyl chain of the gangliosidoide was restricted by the interaction with glycophorin. It can be concluded that GM1 located near glycophorin, a receptor of the virus, interferes with the activity of viral F protein, inhibiting the fusion of liposome to erythrocyte.     

Synthesis of (R)-β-nitro alcohols catalyzed by R-selective hydroxynitrile lyase from Arabidopsis thaliana in the aqueous-organic biphasic system

Both enantiomers of β-nitro alcohols are versatile chiral building blocks. However, their synthesis using enzymes as catalysts has received little attention, with the exception of (S)-β-nitro alcohols produced in a reaction catalyzed by an S-selective hydroxynitrile lyase (HNL) from Hevea brasiliensis (HbHNL). An R-selective HNL containing an α/β-hydrolase fold from the noncyanogenic plant Arabidopsis thaliana (AtHNL) accepts nitromethane (MeNO?) as a donor in a reaction with aromatic aldehydes to yield (R)-β-nitro alcohols (Henry reaction; nitro aldol reaction). This reaction proceeded in an aqueous-organic biphasic system. The organic solvent giving the highest enantioselectivity was n-butyl acetate (AcOBu) with an optimum aqueous phase content of 50% (v/v). This is the first example of the R-HNL-catalyzed synthesis of (R)-β-nitro alcohols.     

Polymorphism of glycophorins in nonhuman primate erythrocytes

Using immunoblotting techniques and polyclonal antisera to human erythrocyte glycophorin, we show that erythrocytes of several species of nonhuman primates, including representatives of anthropoid apes (19 chimpanzees, 3 gorillas, 6 orangutans, and 3 gibbons) and Old World monkeys (3 baboons, 5 rhesus monkeys, and 6 cynomologus macaques), contain human glycophorin-like molecules. Each species displays a unique glycophorin profile; in anthropoid apes the profile is more complex than in Old World monkeys and more similar to that seen in humans. The chimpanzee was the only species in which human -like glycophorin was detected but it differed from its human counterpart in electrophoretic mobility and reaction with M-specific monoclonal antibody. In contrast to humans, highly polymorphic glycophorin profiles were observed in each species of anthropoid apes and three distinct patterns were defined in each. No such polymorphism has been found so far among the Old World monkeys in the limited number of animals studied. The major glycophorins in all species but the chimpanzees failed to react with M- or N-specific monoclonal antibodies, suggesting structural differences from the human within the amino terminal regions. The reaction with the minor glycophorins showed inter- and intraspecies variability. All glycophorins, except -like glycophorin in the chimpanzee, reacted with the antiserum to the carboxyl terminal fragment of human glycophorin, indicating a structural relation to the human in this region. An unexpected correlation was observed, in the chimpanzee, between the patterns of electrophoretically resolved glycophorins and the V-A-B-D blood-group phenotypes, allowing the assignment of each determinant to specific glycophorin bands. The basis for the differences observed between human and nonhuman primate glycophorins is not clear but the possibilities include a common nonpolymorphic ancestor and differences in selective pressures.This research was supported by National Institutes of Health Grant 5 RO1 GM16389.     

A re-examination of the effects of chymotrypsin and trypsin on the erythrocyte membrane surface topology

Silver/Coomassie blue staining of human erythrocyte membrane electrophoretograms permits simultaneous visualization and color differentiation of asialoproteins, sialoglycoproteins and lipids in the same gel. Using this technique evidence is provided that chymotrypsin cleaves glycophorin A as well as band 3. The chymotryptic fragmentation pattern of glycophorin A in situ intact cells was different from that generated by trypsin treatment. Chymotryptic cleavage of band 3 generated two Coomassie blue stained fragments at 62,000 and 38,000 Mr, whereas simultaneous cleavage of glycophorin A dimer and glycophorin A B heterodimer yielded yellow silver stained fragments at 68,000 and 47,000 Mr. Trypsin cleaved glycophorin A dimer (88,000 Mr) and monomer (38,000 Mr) to form membrane associated fragments of Mr = 40,000 and 18,000 respectively.     

Effect of alcohols on the structure of caseins: circular dichroism studies of κ-casein A

The alcohols methanol, ethanol, propan-1-ol and 2,2,2-trifluoroethanol were added to aqueous solution of κ-casein A. Far u.v. circular dichroism spectroscopy revealed a greater proportion of -helix structure in the presence of alcohol; the effectiveness of the alcohol in causing this structural change increased in the order methanol to 2,2,2-trifluoroethanol. No alterations in secondary or tertiary structure were found in κ-casein A on reduction and S-carboxymethylation using near and far u.v.c.d. spectroscopy. The isolated macropeptide section of κ-casein was less sensitive to the addition of alcohols; it appeared that the linkage of macropeptide to the para-κ-casein section affected the case with which solvent induced conformational changes occurred. The conformational changes found in the presence of alcohols could not be correlated simply with the changes in the dielectric constant or with the alcohol-induced collapse of the hairy macropeptide layer of casein micelles or ther precipitation.     

The dual effects of alcohols on the kinetic properties of guinea pig liver cytosolic beta-glucosidase

This report demonstrates the effect of primary alcohols on the kinetic properties of guinea pig liver cytosolic beta-glucosidase. Lineweaver-Burk analyses of the kinetic data revealed a biphasic response; at low concentrations the alcohols increased the Vmax 5--7-fold while at higher concentrations they caused a purely competitive type of inhibition. For example, with n-butyl alcohol, increasing the alcohol's concentration in the assay medium from 0 to 0.14 M (0-1% (v/v)) resulted in a progressive increase in Vmax to a value 7-fold above the basal level without affecting the Km. However, between 0.14 and 0.54 M (1 and 4% (v/v)) n-butyl alcohol, the Km for 4-methylumbelliferyl-beta-D-glucopyranoside increased significantly from 0.14 to 0.93 mM. In contrast to n-butyl alcohol or isobutyl alcohol, which are potent activators, structurally related compounds like sec-butyl alcohol, tert-butyl alcohol, butylurea, and butanesulfonic acid did not stimulate the activity of the cytosolic beta-glucosidase. In the concentration range where activation was observed, conventional secondary replots of 1/delta slope versus 1/[alcohol] yielded perfect straight lines, demonstrating that binding of a single molecule of alcohol to the beta-glucosidase was responsible for the initial phase of activation. Furthermore, the glycohydrolase displayed a propensity to bind the longer chain alcohols, as reflected by the KA (binding constant) values of 555, 146, 34.1, and 7.47 mM for ethanol, n-propyl alcohol, n-butyl alcohol, 1-pentanol, respectively. This phenomenon of nonessential activation by alcohols has led us to speculate on the presence of a physiologic activator for the beta-glucosidase in mammalian tissues which contain this enzyme.     

Molten Globule-Triggered Inactivation of a Thermostable and Solvent Stable Lipase in Hydrophilic Solvents

The use of lipase in hydrophilic solvent is usually hampered by inactivation. The solvent stability of a recombinant solvent stable lipase isolated from thermostable Bacillus sp. strain 42 (Lip 42), in DMSO and methanol were studied at different solvent-water compositions. The enzymatic activities were retained in up to 45% v/v solvent compositions. The near-UV CD spectra indicated that tertiary structures were perturbed at 60% v/v and above. Far-UV CD in methanol indicated the secondary structure in Lip 42 was retained throughout all solvent compositions. Fluorescence studies indicated formations of molten globules in solvent compositions of 60% v/v and above. The enzyme was able to retain its secondary structures in the presence of methanol; however, there was a general reduction in β-sheet and an increase in α-helix contents. The H-bonding arrangements triggered in methanol and DMSO, respectively, caused different forms of tertiary structure perturbations on Lip 42, despite both showing partial denaturation with molten globule formations.     

Enzymatic glucosylation of hydrophobic alcohols in organic medium by the reverse hydrolysis reaction using almond-beta-D-glucosidase

Alkyl beta-D-glucosides were synthesized from D-glucose and alcohols by reverse hydrolysis using the commercially available almond beta-D-glucosidase in 9:1 (v/v) acetonitrile-water medium. The main characteristics of this enzyme-catalyzed glucosylation were established by using 2-hydroxybenzyl alcohol. The reaction is entirely regio- and stereoselective. The solvent plays a fundamental role because, by decreasing the water concentration in the medium, the shift of the reaction equilibrium toward synthesis is realized without using an excessive amount of alcohol. Nevertheless, a minimum amount of water is necessary to maintain the enzyme activity. In contrast to the use of the enzyme in aqueous medium, the pH of the added water in acetonitrile did not influence the synthesis. Using this procedure, we have conducted systematic glucosylation of numerous alcohols and we have investigated enzyme specificity and alcohol reactivity. The enzyme has a pronounced affinity for the alcohols containing a phenyl group, and enantioselectivity for the aglycon is obtained with 1-phenylethyl alcohol. Moreover, by using almond beta-D-glucosidase it was also possible to synthesize alkyl beta-D-galactosides. (c) 1995 John Wiley & Sons, Inc.     

Effect of glucose and long-chain fatty acids on synthesis of long-chain alcohols by Candida albicans

Candida albicans, grown aerobically in glucose-containing media, produced C14, C16 and C18 saturated long-chain alcohols only after the end of exponential growth. Contents of C14 alcohols were always lowest, and C16 and C18 alcohol contents about equal. Contents of all three classes of alcohol increased as the concentration of glucose in aerobic cultures harvested after 168 h incubation was raised from 1.0 to 30.0% (w/v). However, in 168 h anaerobic cultures, greatest long-chain alcohol contents in organisms were obtained using media containing 10% (w/v) glucose. Substituting glucose (10%, w/v) with the same concentration of galactose in aerobic cultures greatly decreased contents of long-chain alcohols, while inclusion of 10% (w/v) glycerol virtually abolished their synthesis. Supplementing anaerobic cultures with odd-chain fatty acids induced synthesis of odd-chain alcohols. Maximum conversion of fatty acid to the corresponding long-chain alcohol was observed with heptadecanoic acid. The effect of glucose on production of heptadecanol from exogenously provided heptadecanoic acid was similar to that observed on synthesis of the three major even-chain alcohols in media lacking a fatty-acid supplement. Cell-free extracts of organisms catalysed in vitro conversion of palmitoyl-CoA to 1-hexadecanol.     

Inhibition of the aminopeptidase from Aeromonas proteolytica by aliphatic alcohols. Characterization of the hydrophobic substrate recognition site.

Seven aliphatic and two aromatic alcohols were tested as reporters of the substrate selectivity of the aminopeptidase from Aeromonas proteolytica (AAP). This series of alcohols was chosen to systematically probe the effect of carbon chain length, steric bulk, and inhibitor shape on the inhibition of AAP. Initially, however, the question of whether AAP is denatured in the presence of aliphatic alcohols was addressed. On the basis of circular dichroism (CD), electronic absorption, and fluorescence spectra, the secondary structure of AAP, with and without added aliphatic alcohols, was unchanged. These data clearly indicate that AAP is not denatured in aliphatic alcohols, even up to concentrations of 20% (v/v). All of the alcohols studied were competitive inhibitors of AAP with K(i) values between 860 and 0.98 mM. The clear trend in the data was that as the carbon chain length increases from one to four, the K(i) values increase. Branching of the carbon chains also increases the K(i) values, but large bulky groups, such as that found in tert-butyl alcohol, do not inhibit AAP as well as leucine analogues, such as 3-methyl-1-butanol. The competitive nature of the inhibition indicates that the substrate and each alcohol studied are mutually exclusive due to binding at the same site on the enzyme. On the basis of EPR and electronic absorption data for Co(II)-substituted AAP, none of the alcohols studied binds to the dinuclear metallo-active site of AAP. Thus, reaction of the inhibitory alcohols with the catalytic metal ions cannot constitute the mechanism of inhibition. Combination of these data suggests that each of these inhibitors bind only to the hydrophobic pocket of AAP and, consequently, block the binding of substrate. Thus, the first step in peptide hydrolysis is the recognition of the N-terminal amino acid side chain by the hydrophobic pocket adjacent to the dinuclear active site of AAP.     

Studies on the interaction of Sendai virus with liposomal membranes. Sendai virus-induced agglutination of liposomes containing glycophorin

Liposomes constituted with the major sialoglycoprotein of human erythrocytes, glycophorin, were used as models for studies on cell-virus interactions. Liposomes composed of egg yolk phosphatidylcholine, cholesterol and glycophorin were found to interact with the paramyxovirus HVJ to form aggregates. The aggregation process was temperature dependent: it was maximal at 0 degrees C and decreased with increase of the incubation temperature. The activity of viral neuraminidase is also temperature dependent, and it increases with increase of the incubation temperature; release of N-acetylneuraminic acid was negligible at 0 degrees C. Shift-up of the incubation temperature immediately cancelled HVJ-induced agglutination of liposomes. Viruses attached to liposomes seemed to be released into the supernatant when the 'virus-liposome' complex formed at 0 degrees C was incubated at 37 degrees C, possibly as a result of breakdown of the 'binding' site by neuraminidase. The characteristics of the interaction of HVJ with liposomes containing glycophorin appeared to be phenomenologically similar to those of HVJ-cell interaction.     

Kinetics of thermal denaturation of met-hemoglobin in perturbed solvent: Relevance of bulk-electrostatic and hydrophobic interactions

We studied the kinetics of the heat denaturation (at 50°C) of met-hemoglobin in the presence of various monohydric alcohols. The denaturation rate was slowed by the presence of small concentrations of methanol and ethanol; in all the other cases, i.e., at high concentrations of methanol and ethanol, and in the whole concentration range studied for iso- and n-propanol, an increase in the denaturation rate was observed. Following a procedure already applied to the study of the effects of the same alcohols on the reaction of hemoglobin with oxygen, we separated the overall observed effects into contributions related to the variation of the bulk dielectric constant of the medium (bulk-electrostatic contributions) and contributions not related to variations in this parameter (nonbulk-electrostatic contributions). For all the alcohols, we found a unique correlation law connecting the above nonbulk-electrostatic contributions with the analogous ones previously reported for the T → R transition of hemoglobin. This fact strongly supports the validity of the procedure used and suggests that nonbulk-electrostatic contributions, relative to these two different processes, have a common background and are univocally determined by the extent of the “solvent perturbation” imposed by the presence of the perturbing cosolvents.     

Effects of alcohol and solvent on the performance of lipase from Candida sp. in enantioselective esterification of racemic ibuprofen

The Candida sp. lipase prepared in our lab was used for the resolution of racemic ibuprofen. In order to study the effects of alcohol and solvent on the performance of Candida sp. lipase in enantioselective esterification of racemic ibuprofen, different alcohols were chosen as acyl acceptors in the same solvent, and identical substrates were used in different solvents. The reactions were performed under controlled water activity, thereby permitting the influences of the alcohols and the solvents to be separated from their ability to strip water from the solid enzyme. The results showed that alcohols and solvents had great effects on the performance of Candida sp. lipase.     

Signal transduction by glycophorin A: role of extracellular and cytoplasmic domains in a modulatable process

Binding of ligands to the extracellular region of the erythrocyte transmembrane protein glycophorin A induces a decrease in membrane deformability. Since the property of membrane deformability is regulated by the skeletal proteins on the cytoplasmic side of the membrane, this suggests that ligand binding may initiate a transmembrane signal. To further study this process, we examined which domains of the extracellular region of glycophorin are involved in signal transduction and whether the cytoplasmic domain of the molecule is necessary for transmitting the signal. Using the ektacytometer, we compared the effect on deformability of four monoclonal antibodies that detect different epitopes on glycophorin A. We found that 9A3 (which recognized the amino terminus of glycophorin) caused a 5.8-fold increase in rigidity, R-10 and 10F7 (which recognized epitopes in the mid-region of the extracellular domain) caused a 10.8-fold increase in rigidity and B14 (which binds to glycophorin close to the membrane) caused a 18-fold increase in rigidity. Further, a direct relationship was observed between the degree of antibody-induced rigidity and the amount of glycophorin A that became associated with the skeletal proteins in a Triton shell assay. In Miltenberger V erythrocytes, which contain a hybrid sialoglycoprotein with no cytoplasmic domain, antibody binding did not induce an increase in rigidity. These results imply that glycophorin A is capable of a modulatable form of transmembrane signaling that is determined by the extracellular domain to which the ligand binds, and the cytoplasmic domain of glycophorin A is crucial for this process.     

Drunken Membranes: Short-Chain Alcohols Alter Fusion of Liposomes to Planar Lipid Bilayers

Although the effects of ethanol on protein receptors and lipid membranes have been studied extensively, ethanol’s effect on vesicles fusing to lipid bilayers is not known. To determine the effect of alcohols on fusion rates, we utilized the nystatin/ergosterol fusion assay to measure fusion of liposomes to a planar lipid bilayer (BLM). The addition of ethanol excited fusion when applied on the cis (vesicle) side, and inhibited fusion on the trans side. Other short-chain alcohols followed a similar pattern. In general, the inhibitory effect of alcohols (trans) occurs at lower doses than the excitatory (cis) effect, with a decrease of 29% in fusion rates at the legal driving limit of 0.08% (w/v) ethanol (IC₅₀ = 0.2% v/v, 34 mM). Similar inhibitory effects were observed with methanol, propanol, and butanol, with ethanol being the most potent. Significant variability was observed with different alcohols when applied to the cis side. Ethanol and propanol enhanced fusion, butanol also enhanced fusion but was less potent, and low doses of methanol mildly inhibited fusion. The inhibition by trans addition of alcohols implies that they alter the planar membrane structure and thereby increase the activation energy required for fusion, likely through an increase in membrane fluidity. The cis data are likely a combination of the above effect and a proportionally greater lowering of the vesicle lysis tension and hydration repulsive pressure that combine to enhance fusion. Alternate hypotheses are also discussed. The inhibitory effect of ethanol on liposome-membrane fusion is large enough to provide a possible biophysical explanation of compromised neuronal behavior.     

Interactions between a membrane sialoglycoprotein and planar lipid bilayers

Summary Bilayer membranes formed from lipids dissolved in decane were exposed to glycophorin, a sialoglycoprotein which had been extracted from human red cell membranes. The interaction with the bilayer produced an increase in the steady state electrical conductance of the membrane proportional to the amount added. Fluctuations in membrane current when the electrical potential difference was constant were observed concommitantly with this increase in membrane conductance. The minimum size of the fluctuations corresponds to a conductance of 10^–10 mho. The increase in conductance as well as the current fluctuations persisted after extensive washout of the chamber containing the protein (cisside). Subsequent addition of lectins (wheat germ agglutinin and phytohemoagglutinin) to the cis-side produced rupture of the membranes, whilst these hemoagglutinins added to the trans-side failed to produce an effect. Measurements of changes in surface potential using K⁺ nonactin as a probe indicated that glycophorin induces a negative surface charge. At high protein concentrations, the magnitude of the induced surface potential became independent of glycophorin concentration. The maximum number of charges introduced onto the membrane under these conditions was 1.4×10⁵/m². Cis (but not trans)-side addition of neuraminidase abolished these charges, indicating that they can be ascribed to the sialic acid residues that the protein bears. These results suggest that glycophorin incorporates into bilayer membranes with its N-terminal end (where the sialic acid and carbohydrates are located) facing the cis-side. Spectrin reversibly lowered the glycophorin-induced membrane conductance when added to the trans-side. Cis-side additions failed to produce an effect. Trypsin present on the trans-side irreversibly lowered the membrane conductance. These results indicate that parts of the glycophorin molecule, probably the C-terminal end, are accessible to reagents in the solution bathing the trans-side of the membrane. Thus glycophorin spans the planar bilayer in much the same way as it spans the red cell membrane.     

The effect of peptide length on the cleavage kinetics of 2-chlorotrityl resin-bound ethers.

Different characteristics of cleavage kinetics of resin-bound amino alcohols and their peptide derivatives were observed in acid containing protic and aprotic solvent mixtures. The hydrolysis reactions are hindered by steric crowding around the cleaving C--O bond and accelerated by the special solvation effect of CF(3)CH(2)OH on the peptide chain as well as the increase of the strength and concentration of the acid. In trifluoroacetic acid containing mixtures, trifluoroacetylation of the peptide alcohols was detected. The appearance of O-trifluoroacetyl serine and threonine derivatives is detected in cleavage mixtures containing trifluoroacetic acid in anhydrous solvent.