α-Chymotrypsin superactivity in aqueous solutions of cationic surfactants

α-Chymotrypsin (α-CT) activity was tested in aqueous media with the following cetyltrialkylammonium bromide surfactants in the series methyl, ethyl, propyl and butyl, different in the head group size, and for the sake of comparison also with the anionic sodium n-dodecyl sulfate and the zwitterionic myristyldimethylammonium propanesulfonate. N-glutaryl-l-phenylalanine p-nitroanilide hydrolysis rate was monitored at surfactant concentration above the critical micellar one. Only some cationic surfactants gave superactivity and the head group size had a major weight. The highest superactivity was measured in the presence of cetyltributylammonium bromide. The effect of both nature and concentration of three different buffers was also investigated. There is a dependence of enzyme superactivity on buffer type. Michaelis–Menten kinetics were found. The binding constants of substrate with micellar aggregates were determined in the used buffers and the effective improvement of reaction rate (at the same free substrate concentration in the medium) was calculated. k_cat significantly increased while K_m was little changed after correction to free substrate concentration. The ratio of k_cat to K_m was between 12 and 35 times higher than in pure buffer, depending on buffer and surfactant concentrations. The increase of α-CT activity (30%) was less important in the presence of 1×10⁻² M tetrabutylammonium bromide, a very hydrophobic salt, unable to micellise. Fluorescence spectra showed differences of enzyme conformation in the presence of various surfactants.     

Distinct effect of a cationic surfactant on the transient and steady state phases of 2-naphthyl acetate hydrolysis catalyzed by α-chymotrypsin

Results obtained on the effect of addition of dodecyltrimethylammonium bromide (DTAB) upon the α-chymotrypsin (α-CT) catalyzed hydrolysis of 2-naphthyl acetate (2-NA) under steady state conditions for the acyl–enzyme intermediate are compared with those previously obtained in the transient (pre-steady state or “burst”) phase. It is found that, while in the transient phase there is no effect of DTAB addition on the kinetic parameters at concentrations below the critical micelle concentration (CMC) of the surfactant, super-activity is observed when the acyl–enzyme intermediate reaches the steady state condition. This difference implies that the surfactant does not modify either the formation or the decomposition of the enzyme–substrate complex (transient phase) but notably increases the rate of disruption of the acyl–enzyme intermediate.     

Lysine acetylation can generate highly charged enzymes with increased resistance toward irreversible inactivation

This paper reports that the acetylation of lysine ε-NH₃⁺ groups of α-amylase—one of the most important hydrolytic enzymes used in industry—produces highly negatively charged variants that are enzymatically active, thermostable, and more resistant than the wild-type enzyme to irreversible inactivation on exposure to denaturing conditions (e.g., 1 h at 90°C in solutions containing 100-mM sodium dodecyl sulfate). Acetylation also protected the enzyme against irreversible inactivation by the neutral surfactant TRITON X-100 (polyethylene glycol p-(1,1,3,3-tetramethylbutyl)phenyl ether), but not by the cationic surfactant, dodecyltrimethylammonium bromide (DTAB). The increased resistance of acetylated α-amylase toward inactivation is attributed to the increased net negative charge of α-amylase that resulted from the acetylation of lysine ammonium groups (lysine ε-NH₃⁺ → ε-NHCOCH₃). Increases in the net negative charge of proteins can decrease the rate of unfolding by anionic surfactants, and can also decrease the rate of protein aggregation. The acetylation of lysine represents a simple, inexpensive method for stabilizing bacterial α-amylase against irreversible inactivation in the presence of the anionic and neutral surfactants that are commonly used in industrial applications.     

Effects of cationic micelles on the rate of reaction of p-nitrophenyl esters with dihydrolipoic acid and dihydrolipoylamido derivatives

Dihydrolipoic acid (DHLA) 2a and its surfactant derivatives, trialkyl(2-lipoylamidoethyl) ammonium salts 2b-c, have been investigated, mainly in micellar solutions of cetyltrimethyl-ammonium bromide (CTABr), as esterolytic reagents toward p-nitrophenyl esters. The origins of the observed kinetic effects are discussed, and the reactivity of these reagents are compared with that of other thiolytic systems. The results indicate that DHLA, although not a surfactant, is effectively comicellized by CTABr, and micelles of CTABr and DHLA are among the most effective esterolytic systems, at moderately alkaline pHs, so far reported.     

Regulation of catalytic properties of enzymes in "inverse micelles"]

A triple system (inverse micellae) that simulates the membrane environment of the enzyme was studied. Inverse micellae were obtained using anionic (aerosol OT), synthetic (Brij 56), and natural (lecithin) surfactants. It was found that upon inclusion of an enzyme into inverse micellae, its activity can be regulated by changing the structure and nature of the surfactant matrix. It was shown that enzyme activity in micellar environment is much higher than in water solution. Moreover, the enzyme solubilized in inverse micellae (acid phosphatase) shows a superactivity. It was found that surfactants specifically interact with solubilized enzyme, and the activity of the enzyme is inversely proportional to surfactant concentration. The mechanisms of viscotropic regulation of enzyme activity are discussed.     

The phenomenon of super activity in dihydrofolate reductase entrapped inside reverse micelles in apolar solvents

Bovine liver dihydrofolate reductase has been solubilized in reverse micelles of cationic surfactant cetyltrimethylammonium bromide (CTAB) in isooctane-chloroform (1:1,V/V) mixture. Variation of waterpool (WO), pH and surfactant concentration showed that the enzyme activity was regulated by these parameters and was higher than the activity found in aqueous buffer (defined as superactivity); the maximum being at WO 13.3, pH 7.0 and CTAB concentration 75 mM. The Michaelis constants, Km for the substrate FAH2 and NADPH were found to be greater than those determined in water. Since reverse micelles have some features similar to those of biomembranes, display of super activity by dihydrofolate reductase indicates that enzymes in vivo may possess higher activity than actually observed in vitro studies in aqueous solutions.     

The improvement of chloroperoxidase activities in the presence of ammonium salts and cationic surfactants

The catalytic activities of chloroperoxidase (CPO) including halogenation, oxidation, and peroxidation were investigated in the presence of ammonium salts: tetramethylammonium bromide (TMABr), tetraethylammonium bromide (TEABr), tetrapropylammonium bromide (TPABr), tetrabutylammonium bromide (TBABr), and cationic surfactants: dodecyltrimethylammonium bromide (DTABr) and cetyltrimethylammonium bromide (CTABr). All the mentioned activities were promoted in most cases. The highest modified activity (ABTS peroxidation) was 18.16 times higher in the presence of TMABr than that in pure buffer. The activity enhancement was strongly dependent on the concentration and the hydrophobic chain length of additives, and the structure of substrates. The kinetic parameters showed that the activation was mainly attributed to an increase in k_cat due probably to a catalytically favorable conformation of CPO induced by the additives. Moreover, a lower K_m and higher ratio of k_cat/K_m (specificity constant) was obtained, indicating that both the affinity and specificity of CPO to substrates were improved in the presence of additives. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Cross talk between protein kinase CK2 and eukaryotic translation initiation factor eIF2β subunit

The β-subunit of eukaryotic translation initiation factor eIF2 is a substrate and a partner for protein kinase CK2. Surface plasmon resonance analysis shows that the truncated form corresponding to residues 138–333 of eIF2β (eIF2β-CT) interacts with CK2α as efficiently as full length eIF2β, whereas the form corresponding to residues 1–137, which contains the CK2 phosphorylation sites, (eIF2β-NT) does not bind. The use of different mutants and truncated forms of CK2α allowed us to map the basic segment K74–K83 at the beginning of helix αC and residues R191R195K198 in the p+1 loop as the main determinants for the binding to eIF2β-CT of either the isolated CK2α subunit or the CK2 holoenzyme. The presence of eIF2β-CT stimulated the activity of CK2α towards the RRRAADSDDDDD peptide substrate; effect that was not observed with the CK2α K74-77A whose ability to bind to eIF2β-CT is severely impaired. Gel filtration analysis confirmed the ability of CK2α to form complexes with eIF2β-CT, and the contribution of the basic cluster in CK2α (K74–K77) in this association.     

Effect of various surfactants (cationic,anionic and non-ionic) on the growth of <Emphasis Type="Italic">Aspergillus parasiticus</Emphasis> (NRRL 2999) in relation to aflatoxin production

The effect of surfactants (two cationic, one anionic and three non-ionic) at 0.001, 0.01, 0.1 and 1.0 % concentrations on aflatoxin production, ergosterol content and sugar consumption by Aspergillus parasiticus (NRRL 2999) in YES liquid culture medium is reported. At 0.01% concentration, the cationic surfactants, cetyl dimethyl ammonium bromide (CDAB) and dodecyl trimethyl ammonium bromide (DTAB), and the anionic surfactant, sodium dodecyl sulfate (SDS), completely inhibited spore germination, while DTAB also inhibited the production of ergosterol and toxin (p < 0.05). At a concentration of 0.001%, CDAB was found to enhance toxin production, while SDS was found to inhibit it when compared with other surfactants. Non-ionic surfactants, polyoxyethylene sorbitan monolaurate (Tween-20), polyoxyethylene lauryl ether (Brij-35) and ethoxylated p-tert-octylphenol (Triton X-100) delayed the spore germination up to day 5 at all concentrations and inhibited toxin and ergosterol production at 0.001% concentration. The affect was found to be dose-dependent from 0.001% to 1%, for Triton X-100 only. Positive correlation between ergosterol content and toxin production in the presence of different surfactants at various time periods (3, 5, 7, 9 and 12 days) was found. Tween-20 was most effective in inhibiting toxin production on day 7, when aflatoxin production was found to be maximal in control group. Sugar consumption was directly proportional to the ergosterol content, showing a significant correlation with aflatoxin production.     

On the meaning of enzymatic superactivity in reverse micelles

Summary Two types of superactivity can be defined. One, is with respect to the activity for a fixed average substrate concentration in the water pool; the pushing of the charged substrate by the likewise charged micellar surface is responsible for the superactivity and its bell-shaped dependence on the hydration ratio. The other, is with respect to the activity in a bulk aqueous solution having a substrate concentration equal to a fixed overall concentration [S^–]_ov in the entire reverse micellar solution. In this case, the pushing effect, the constraint of a fixed [S^–]_ov and the partitioning of the substrate in the surfactant layer are responsible for the bell-shaped dependence. Superactivity exists for low substrate partitioning in the surfactant layer, subactivity for high partitioning.     

Effect of surfactant solubilization on biodegradation of polychlorinated biphenyl congeners by Pseudomonas LB400

A variety of commercial surfactants were tested to determine their effect on polychlorinated biphenyl (PCB) transformation by Pseudomonas LB400. Initial tests determined that most surfactants were fully or partially able to solubilize the PCB congeners 2,5,2′-chlorobiphenyl (CBP), 2,4,2′,4′-CBP, 2,3,5,2′,5′-CBP and 2,4,5,2′,4′,5′-CBP, at concentrations above the surfactants' critical micelle concentration (CMC). Surfactants were also found to have no negative effect on bacterial survival, as cell numbers were the same or higher after incubation in the presence of surfactants than after incubation without surfactants. A comparison of the extent of biotransformation of single PCB congeners by the bacterium revealed that, at surfactant concentrations above the CMC, the presence of an anionic surfactant promoted while nonionic surfactants inhibited PCB transformation, compared to a control with no surfactant. The rates of transformation of PCB congeners were also higher in the presence of the anionic surfactant compared to the control. The inhibitory effects of a nonionic surfactant, Igepal CO-630 at a concentration above its CMC, on transformation of 2,4,5,2′,5′-CBP could be eliminated by diluting the surfactant/PCB solution to a concentration close to the surfactant CMC. Received: 26 October 1998 / Received revision: 5 March 1999 / Accepted: 14 March 1999     

Enhanced enzymatic activity in reverse micelles

Summary The bell shaped dependence of the superactivity of enzymes solubilized in ionic reverse micelles (RMs) on the hydration ratio (W₀) is theoretically explained. The superactivity is due to enhanced concentration of the substrate (which has the same kind of charge as that of the surfactant head groups) near the enzyme surface. The opposing effects of the increase in the absolute charge of the surface of the RM and in the water pool width with W₀ cause a maximum in activity.     

Enzymatic synthesis of on the preparative scale

The problems inherent in the enzymatic and chemical synthesis of (SAM) led us to develop an efficient, simple method for the synthesis of large amounts of labeled SAM. Previously, we reported that the problem of product inhibition of E. coli SAM synthetase encoded by the metK gene was successfully overcome in the presence of sodium p-toluenesulfonate (pTsONa). This research has now been expanded to demonstrate that product inhibition of this enzyme can also be overcome by adding a high concentration of β-mercaptoethanol (βME), acetonitrile, or urea. In addition, a recombinant strain of E. coli has been constructed that expresses the yeast SAM synthetase encoded by the sam2 gene. The yeast enzyme does not have the problem of product inhibition seen with the E. coli enzyme. Complete conversion of 10 mM methionine to SAM was achieved in incubations with either the recombinant yeast enzyme and 1 molar potassium ion or the E. coli enzyme in the presence of additives such as βME, acetonitrile, urea, or pTsONa. The recombinant yeast SAM synthetase was used to generate SAM in situ for use in the multi-enzymatic synthesis of precorrin 2.     

Formation of Dynamic Soluble Surfactant-induced Amyloid β Peptide Aggregation Intermediates

Intermediate amyloidogenic states along the amyloid β peptide (Aβ) aggregation pathway have been shown to be linked to neurotoxicity. To shed more light on the different structures that may arise during Aβ aggregation, we here investigate surfactant-induced Aβ aggregation. This process leads to co-aggregates featuring a β-structure motif that is characteristic for mature amyloid-like structures. Surfactants induce secondary structure in Aβ in a concentration-dependent manner, from predominantly random coil at low surfactant concentration, via β-structure to the fully formed α-helical state at high surfactant concentration. The β-rich state is the most aggregation-prone as monitored by thioflavin T fluorescence. Small angle x-ray scattering reveals initial globular structures of surfactant-Aβ co-aggregated oligomers and formation of elongated fibrils during a slow aggregation process. Alongside this slow (minutes to hours time scale) fibrillation process, much faster dynamic exchange (k_ex ∼1100 s⁻¹) takes place between free and co-aggregate-bound peptide. The two hydrophobic segments of the peptide are directly involved in the chemical exchange and interact with the hydrophobic part of the co-aggregates. Our findings suggest a model for surfactant-induced aggregation where free peptide and surfactant initially co-aggregate to dynamic globular oligomers and eventually form elongated fibrils. When interacting with β-structure promoting substances, such as surfactants, Aβ is kinetically driven toward an aggregation-prone state.     

Stabilization of Crystalline Acid Carboxypeptidase from Penicillium janthinellum by Nonionic Surfactants,and Inhibition of Enzyme Activity by Anionic Compounds

The crystalline acid carboxypeptidase from Penicillium janthinellum IFO-8070 was stabilized by the addition of nonionic surfactants, such as Triton X-100, Brij 35, Span 40, and Tween 20. In the presence of these stabilizers, extremely diluted enzyme (0.3 μg/ml of 50 mm sodium acetate buffer, pH 3.7) was almost completely stable after 2 days incubation at 25°C. About 35% and 20% of the enzyme activities were activated by the addition of Triton X-100 and Brij 35, respectively. Triton X-100 completely retarded inactivation at freezing (?15°C). On the other hand, anionic surfactants of SLS and LBSA, and cationic surfactant of cetyltrimethylammonium bromide strongly inactivated the enzyme. The inhibition of the fatty acid series was roughly proportional to the molecular weight of the inhibitor. Di-, and Tri-carboxylic acids also inhibited the enzyme activity.     

Enantioselective ester hydrolase from Sphingobacterium sp. 238C5 useful for chiral resolution of β-phenylalanine and for its β-peptide synthesis

A novel enzyme, β-phenylalanine ester hydrolase, useful for chiral resolution of β-phenylalanine and for its β-peptide synthesis was characterized. The enzyme purified from the cell free-extract of Sphingobacterium sp. 238C5 well hydrolyzed β-phenylalanine esters (S)-stereospecifically. Besides β-phenylalanine esters, the enzyme catalyzed the hydrolysis of several α-amino acid esters with l-stereospecificity, while the deduced 369 amino acid sequence of the enzyme exhibited homology to alkaline d-stereospecific peptide hydrolases from Bacillus strains. Escherichia coli transformant expressing the β-phenylalanine ester hydrolase gene exhibited an about 8-fold increase in specific (S)-β-phenylalanine ethyl ester hydrolysis as compared with that of Sphingobacterium sp. 238C5. The E. coli transformant showed (S)-enantiomer specific esterase activity in the reaction with a low concentration (30 mM) of β-phenylalanine ethyl ester, while it showed both esterase and transpeptidase activity in the reaction with a high concentration (170 mM) of β-phenylalanine ethyl ester and produced β-phenylalanyl-β-phenylalanine ethyl ester. This transpeptidase activity was useful for β-phenylalanine β-peptide synthesis.     

Binding of cationic surfactants to DNA, protein and DNA-protein mixtures

Extent of binding (gamma 2(1)) of cationic surfactants cetyltrimethyl ammonium bromide (CTAB), myristyltrimethyl ammonium bromide (MTAB) and dodecyl trimethyl ammonium bromide (DTAB) to calf-thymus DNA, bovine serum albumin (BSA) and to their binary mixture respectively have been measured as function of bulk concentration of the surfactant by using equilibrium dialysis technique. Binding of CTAB has been studied at different pH, ionic strength (mu), temperature and biopolymer composition and with native and denatured states of the biopolymers. The chain-length of different long chain amines plays a significant role in the extent of binding under identical solution condition. The binding ratios for CTAB to collagen, gelatin, DNA-collagen and DNA-gelatin mixtures respectively have also been determined. The conformational structures of different biopolymers are observed to play significant role in macromolecular interactions between protein and DNA in the presence of CTAB. From the experimental values of the maximum binding ratio (gamma 2m) at the saturation level for each individual biopolymer, ideal values (gamma 2m)id have been theoretically calculated for binary mixtures of biopolymers using additivity rule. The protein-DNA-CTAB interaction in mixture has been explained in terms of the deviation (delta) of (gamma 2m) from (gamma 2m)id in the presence of a surfactant in bulk. The binding of surfactants to biopolymers and to their binary mixtures are compared more precisely in terms of the Gibbs' free energy decrease (-delta G degree) for the saturation of the binding sites in the biopolymers or biopolymer mixtures with the change of the bulk surfactant activity from zero to unity in the rational mole fraction scale.     

Ca3(PO4)2:Eu3+ phosphor preparation with different morphologies and their fluorescence properties

Ca₃(PO₄)₂:Eu³⁺ phosphor was prepared using a facile chemistry method in the presence of surfactants. The effects of surfactants on the morphology and photoluminescence properties of Ca₃(PO₄)₂:Eu³⁺ phosphor were investigated. The morphology of the phosphor was significantly influenced by the surfactants employed. When nonionic surfactant glyceryl monostearate and anionic surfactant sodium dodecylbenzene sulfonate were employed, the phospor powders are composed of a large number of homogeneous spherical particles with sizes of 0.3–0.6 µm and 2–3 µm, respectively. By contrast, when cationic surfactant cetyltrimethylammonium bromide was used, the morphology of the phosphor is completely different. The product is an excellent cuboid, and the phosphor prepared with 2.5 mmol cetyltrimethylammonium bromide showed higher luminescent intensity than phosphors prepared with the other two types of surfactants. Copyright © 2013 John Wiley & Sons, Ltd.     

Enzymatic synthesis of trisaccharides and alkyl β-d-glucosides by the transglycosylation reaction of β-glucosidase from Fusarium oxysporum

Purified β-glucosidase from Fusarium oxysporum catalyses hydrolysis and transglycosylation reactions. By utilizing the transglycosylation reaction, trisaccharides and alkyl β-d-glucosides were synthesized under optimal conditions in the presence of various disaccharides and alcohols. The yields of trisaccharides and alkyl β-d-glucosides were 22–37% and 10–33% of the total sugar, respectively. The enzyme retained 70–80% of its original activity in the presence of 25% (w/v) methanol, ethanol and propanol. Thus, β-glucosidase from F. oxysporum appears to be an ideal enzyme for the synthesis of useful trisaccharides and alkyl β-d-glucosides.     

Responsiveness of κ-carrageenan microgels to cationic surfactants and neutral salts

The objective of this study was to examine the responsiveness of chemically cross-linked κ-carrageenan microspheres to different types of neutral salt electrolytes as well as to surfactants of varying chain lengths. In the presence of increasing salt concentration microsphere size changed radically from D[4,3] values of 320 μm to approximately 160 μm. The level of salt concentration needed to bring about this change varied depending on electrolyte type. This common behaviour was attributed to the difference in free cationic counter-ions concentration between the inside and outside of the microsphere and can be explained due to the effect of the Donnan equilibrium. The rheological properties of these microgels in their swollen and collapsed states were also explored with results showing that the collapsed microspheres had a greater impact on the viscosity of the system probably as a result of some aggregation of the collapsed microgels at rest due to surface charge screening at these high salt concentrations. The effect of surfactant on microsphere size showed a dramatic drop in D[4,3] values from 320 μm to approximately 120 μm for BAC, DoTAB, MTAB and CTAB at specific critical concentrations. This critical aggregation concentration was found to increase linearly on a log–log scale with the critical micelle concentration of these surfactants in water, indicating that the alkyl chain length of the surfactants had an effect on the critical aggregation concentration.