Activity Of Chromobacterium Viscosum Lipase Research Articles

Hydrophobically Tailored Carbon Dots toward Modulating Microstructure of Reverse Micelle and Amplification of Lipase Catalytic Response.

This article delineates the modulation of microstructure of cationic reverse micelle utilizing hydrophobically modified carbon dots (CDs) with varying surface functionalizations. Citric acid was used as the source of the carbon core, and Na-salt of glycine, glycine, Na-salt of 11-aminoundecanoic acid, 11-aminoundecanoic acid, and n-hexadecylamine were used for the surface fabrication of CDs to produce CD 1s, CD 1a, CD 2s, CD 2a, and CD 3, respectively. All these CDs having dimension of 5-7 nm were characterized by spectroscopic and microscopic techniques. The hydrodynamic diameter of cetyltrimethylammonium bromide (CTAB) reverse micelle (CTAB/isooctane/n-hexanol/water) at z ([cosurfactant]/[surfactant]) = 6.4 and W0 ([water]/[surfactant]) = 44 is around 15-20 nm. Interestingly, the size of the water-in-oil (w/o) microemulsions dramatically increased up to 120-200 nm upon doping hydrophobic surface functionalized CD 2a and CD 3. This is possibly due to change in the micellar exchange dynamics and reorganization of the micellar aggregates via hydrophobic interaction between surfactant (CTAB) tail and hydrophobic surface modifier of the carbon dots. However, no alteration in the size of reverse micelles was noted in the presence of carbon dots CD 1s, CD 1a, and CD 2s. Spectroscopic and microscopic investigations confirmed that the hydrophobic CD 2a and CD 3 were localized at the interface of reverse micelles whereas CD 1s, CD 1a, and CD 2s were possibly located in the water pool (away from interface). The activity of Chromobacterium viscosum lipase encapsulated within CD 3 and CD 2a doped significantly large CTAB reverse micelles showed remarkable improvement (3.7-fold and 3.4-fold) in its catalytic response. However, hydrophilic carbon dots CD 1s and CD 2s as well as moderately hydrophobic CD 1a had no significant effect on the microstructure of reverse micelles as well as on the lipase activity.

Nonionic Surfactants: A Key to Enhance the Enzyme Activity at Cationic Reverse Micellar Interface

The primary objective of the present study is to understand how the different nonionic surfactants modify the anisotropic interface of cationic water-in-oil (W/O) microemulsions and thus influences the catalytic efficiency of surface-active enzymes. Activity of Chromobacterium viscosum lipase (CV-lipase) was estimated in several mixed reverse micelles prepared from CTAB and four different nonionic surfactants, Brij-30, Brij-92, Tween-20, and Tween-80/water/isooctane/n-hexanol at different z ([cosurfactant]/[surfactants]) values, pH 6 (20 mM phosphate), 25 degrees C across a varying range of W0 ([water]/[surfactants]) using p-nitrophenyl-n-octanoate as the substrate. Lipase activity in mixed reverse micelles improved maximum up to approximately 200% with increasing content of non-ionic surfactants compared to that in CTAB probably due to the reduced positive charge density as well as plummeted n-hexanol (competitive inhibitor of lipase) content at the interfacial region of cationic W/O microemulsions. The highest activity of lipase was observed in CTAB (10 mM) + Brij-30 (40 mM)/isooctane/n-hexanol)/water system, k2 = 913 +/- 5 cm3 g-1 s-1. Interestingly, this observed activity is even higher than that obtained in sodium bis (2-ethyl-1-hexyl) sulfosuccinate (AOT)/n-heptane reverse micelles, the most popular W/O microemulsion in micellar enzymology. To ascertain the influence of non-ionic surfactants in improving the activity of surface-active enzymes is not limited to lipase only, we have also investigated the catalytic activity of Horseradish peroxidase (HRP) in different mixed W/O microemulsions. Here also following the similar trend as observed for lipase, HRP activity enhanced up to 2.5 fold with increasing concentration of nonionic surfactants. Finally, the enzyme activity was correlated with the change in the microenvironment of mixed reverse micelles by steady-state fluorescence study using 8-anilino-1-napthalenesulphonic acid (ANS) as probe.

Lipase-catalyzed esterification of fatty acid in DMSO (dimethyl sulfoxide) modified AOT reverse micellar systems

AOT reverse micellar system was modified with DMSO for improved esterification activity of Chromobacteriumviscosum lipase (glycerol–ester hydrolase, EC 3.1.1.3). The enzymatic activity was strongly affected by the concentration of DMSO, and maximum activity was obtained at 30–40 mM. The various relevant physical parameters such as w0 (molar ratio of water to AOT), pH and reaction temperature that influence the activity of lipase were studied in order to obtain the best value and compared with those in simple AOT reverse micelles. The apparent activation energy decreased in the presence of DMSO. The stability of lipase entrapped in modified AOT systems was excellent, and the half-life was about 3.25 times than that observed in simple AOT systems at 25°C. A simple first-order deactivation model was considered to determine the deactivation rate constant. The thermodynamic stability of lipase in reverse micelles was measured by the Gibbs free energy. A fluorescence study was performed to provide information on structural changes in AOT reverse micelles which was accompanied by the addition of DMSO.

Activity of acetone-treated Chromobacterium viscosum lipase in AOT reverse micelles in the presence of low molecular weight polyethylene glycol

The activity of Chromobacterium viscosum lipase (glycerol-ester hydrolase, EC 3.1.1.3) entrapped in AOT/isooctane/water reverse micelles was significantly enhanced by pretreatment with acetone, using the hydrolysis of olive oil as a model reaction. The activity of acetone treated lipase was further enhanced when low molecular weight polyethylene glycol (PEG 400) was introduced into AOT reverse micelles. To know the effects of acetone treatment on the lipase activity in simple AOT and mixed AOT/PEG 400 reverse micelles, the influence of various parameters, such as W 0 (molar ratio of water to surfactant), pH, surfactant concentration, ionic strength and reaction temperature were investigated and compared with those for native lipase in simple AOT reverse micelles. The optimal activities of treated lipase in AOT reverse micelles with and without PEG 400 were 2.0 and 1.6 times higher respectively than that of native lipase in AOT reverse micelles. A kinetic model that considers substrate adsorption equilibrium between the bulk phase of organic solvent and the micellar phase was successfully used to understand the improvement of the lipase activity. The Michaelis constant ( K m) and substrate adsorption equilibrium constant (K ad ) were obviously reduced compared with those for native lipase in AOT reverse micelles. The stability of the lipase in reverse micelles was also studied, and the values of half-life time ( t 1/2) were determined from residual activity profiles.

Surfactant tail length-dependent lipase activity profile in cationic water-in-oil microemulsions

The catalytic activity of Chromobacterium viscosum lipase (CV-lipase) was estimated across varying surfactant tail lengths (C-10–C-18) in water-in-oil (w/o) microemulsions of cationic surfactants containing four different hydroxyethyl-substituted head groups. An attempt to find a correlation, if any, between the activity of interfacially solubilized lipase and the varying surfactant tails was made for the first time in micellar enzymology. The second-order rate constant, k 2 , in lipase-catalyzed hydrolysis of p-nitrophenyl- n-hexanoate at pH 6.0 and 25 °C shows an improvement in enzyme activity (∼30–140%) across different head groups of amphiphiles with increasing tail lengths in varying solution compositions. Improvement of enzyme activity is prominent in ascending from C-10 to C-14/C-16, depending on the nature of polar head group. The hydrolytic activity of lipase in different surfactant (50 mM)/water/isooctane/ n-hexanol with varying z= [alcohol]/[surfactant] (6.4 or 4.8) was amplified by 25–250% with increment in surfactant tail length in comparison with widely used cationic w/o microemulsions having solution compositions ( z = 16 ) . As a notable outcome of this research, we found w/o microemulsions of 25 mM tetradecyltrimethylammonium bromide/water/isooctane/ n-hexanol ( z = 8 ) producing the highest ever activity of lipase in any w/o microemulsions.

Pretreatment ofChromobacterium viscosum lipase with acetone increases its activity in sodium bis-(2-ethylhexyl) sulfosuccinate (AOT) reverse micelles

The activity of Chromobacterium viscosum lipase for hydrolysis of olive oil in sodium bis-(2-ethylhexyl) sulfosuccinate (AOT) reverse micelles was increased by pretreatment with acetone. In contrast to the untreated lipase, no sharp fall in the activity of the treated lipase at higher W0 (water to AOT molar ratio) values was observed. The fluorescence emission intensity of the treated lipase in reverse micelles was higher than that of the untreated lipase but the maximal emission wavelength (λmax) was the same for both lipases. A kinetic model that considers the free substrate in equilibrium with the substrate adsorbed on the micellar surface was successfully used to better understand the activity enhancement. The Michaelis constant (Km) and substrate adsorption equilibrium constant (Kad) were reduced by lipase pretreatment with acetone whereas the maximum reaction rate (vmax) remained unaltered. Copyright © 2005 Society of Chemical Industry

Improving the Lipase Activity Profile in Cationic Water-in-Oil Microemulsions of Hydroxylated Surfactants

The activity of Chromobacterium viscosum lipase (CV-lipase) in cationic water-in-oil (w/o) microemulsions is significantly lower compared to that in bis(2-ethylhexyl)sulfosuccinate sodium salt (AOT)-based (anionic) systems.26,36,38,39 In the present study, we estimated the second-order rate constants k2 in lipase-catalyzed hydrolysis of p-nitrophenylcaproate, in newly developed cationic w/o microemulsions of synthesized surfactants (2−6, Chart 1) containing hydroxyethyl moieties at the polar headgroup. The kinetic studies at pH = 6.0 (pH refers to the pH of the aqueous buffer solutions used in preparing the w/o microemulsions) show that the catalytic efficiency of CV-lipase was systematically increased with the sequential increment of hydroxyethyl groups at the polar heads of surfactants (1−4, Chart 1), possibly due to the increase in the interfacial concentration of water [H2O]i in consequence of the added hydrogen bonding ability of hydroxyl groups. To this end, we found that in a 0.05 M 4/water/32.3:1 ...

A kinetic model for enzymatic reactions in reverse micellar systems involving water-insoluble substrates and enzyme activators

The activity of Chromobacterium viscosum lipase (glycerol-ester hydrolase, EC 3.1.1.3) entrapped in AOT/isooctane reverse micelles was significantly increased by the addition of short chain polyethylene glycols (PEGs) or methoxypolyethylene glycols (MPEGs) for the hydrolysis of olive oil. To understand enzyme activity in the presence of PEG 400 or MPEG 550 molecules, a kinetic model was proposed. The validity of this model was verified by experimental data on the lipase-catalyzed hydrolysis of olive oil in AOT/isooctane reverse micellar systems, in which PEG 400 or MPEG 550 had been added. The large value of the equilibrium constant (kD) for enzyme activation indicated that the affinity between C viscosum lipase and PEG 400 or MPEG 550 molecules was very strong. The Michaelis constant (Km) predicted by the proposed model explained enzymatic reactions more exactly than that by the previously published model. Copyright © 2003 Society of Chemical Industry

Increased activity of Chromobacterium viscosum lipase in AOT reverse micelles in the presence of short chain methoxypolyethylene glycol

AbstractThe activity of Chromobacterium viscosum lipase (glycerol‐ester hydrolase, EC 3.1.1.3) entrapped in AOT/isooctane and AOT/Tween 85/isooctane reverse micelles was significantly increased by the addition of short chain methoxypolyethylene glycols (MPEGs), taking the hydrolysis of olive oil as a model reaction. The molecular weight of MPEG had a strong effect on the lipase activity, and MPEG of nominal molecular weight 550 was found to be the most effective. To optimize the factors affecting enzymatic hydrolysis of olive oil in reverse micellar systems containing MPEG 550, the effect of various parameters, such as Wo (molar ratio of water to surfactant), pH, ionic strength, surfactant concentration and temperature were investigated. A kinetic model considering the substrate adsorption equilibrium between the bulk phase of organic solvent and the micellar phase was also successfully used to understand the enzyme activity in the presence of MPEG 550. Both the Michaelis constant and the substrate adsorption equilibrium constant were obviously reduced as compared with those obtained in the simple AOT reverse micellar system.© 2001 Society of Chemical Industry

Kinetics and stability of a chromobacterium viscosum lipase in reversed micellar and aqueous media

The lipolytic activity of Chromobacterium viscosum lipase B (EC 3.1.1.3.; triacylglycerol hydrolase) solubilized both in water and AOT/isooctane reversed micelles has been investigated using triolein as a substrate. The influence of relevant parameters in the catalytic activity such as temperature, pH, surfactant and substrate concentrations, and water content was tested and compared in both media. A study of stability of the lipase was carried out, with particular reference to the influence of pH. Three major effects of the encapsulation of the lipase in the micelles were observed: increased activity (up to 7 times higher than in water), greater stability, specially at pH 7, and higher resistance to thermal deactivation.