Activity and stability of yeast alcohol dehydrogenase (YADH) entrapped in aerosol OT reverse micelles

The activity and stability of yeast alcohol dehydrogenase (YADH) entrapped in aerosol OT reverse micellar droplets have been investigated spectrophotometrically. Various physical parameters, e.g., water pool size, w(0), pH, and temperature, were optimized for YADH in water/AOT/isooctane reverse micelles. It was found that the enzyme exhibits maximum activity at w(0) = 28 and pH 8.1. It was more active in reverse micelles than in aqueous buffers at a particular temperature and was denatured at about 307deg;C in both the systems. At a particular temperature YADH entrapped in reverse micelles was less stable than when it was dissolved in aqueous buffer.     

Stability and activity of alcohol dehydrogenases in W/O-microemulsions: enantioselective reduction including cofactor regeneration

Microemulsions provide an interesting alternative to classical methods for the conversion of less water-soluble substrates by alcohol dehydrogenase, but until now stability and activity were too low for economically useful processes. The activity and stability of the enzymes are dependent on the microemulsion composition, mostly the water and the surfactant concentration. Therefore, it is necessary to know the exact phase behavior of a given microemulsion reaction system and the corresponding enzyme behavior therein. Because of their economic and ecologic suitability polyethoxylated fatty alcohols were investigated concerning their phase behavior and their compatibility with enzymes in ternary mixtures. The phase behavior of Marlipal O13-60 (C13EO6 in industrial quality)/cyclohexane/water and its effect on the activity and stability of alcohol dehydrogenase from Yeast (YADH) and horse liver (HLADH) and the carbonyl reductase from Candida parapsilosis (CPCR) is presented in this study. Beside the macroscopic phase behavior of the reaction system, the viscosity of the system indicates structural changes of aggregates in the microemulsion. The changes of the enzyme activities with the composition are discussed on the basis of transitions from reverse micelles to swollen reverse micelles and finally, the transition to the phase separation. The formate dehydrogenase from Candida boidinii was used for the NADH-regeneration during reduction reactions. While the formate dehydrogenase did not show any kinetic effect on the microemulsion composition, the other enzymes show significant changes of activity and stability varying the water or surfactant concentration of the microemulsion. Under certain conditions, stability could be maintained with HLADH for several weeks. Successful experiments with semi-batch processes including cofactor regeneration and product separation were performed.     

Enzymatic redox cofactor regeneration in organic media: functionalization and application of glycerol dehydrogenase and soluble transhydrogenase in reverse micelles

An enzymatic system for the regeneration of redox cofactors NADH and NADPH was investigated in nanostructural reverse micelles using bacterial glycerol dehydrogenase (GLD) and soluble transhydrogenase (STH). Catalytic conversion of NAD+ to NADH was realized in the sodium dioctylsulfosuccinate (AOT)/isooctane reverse micellar system harboring GLD and a sacrificial substrate, glycerol. The initial rate of NADH regeneration was enhanced by exogenous addition of ammonium sulfate into the reverse micelles, suggesting that NH4+ acts as a monovalent cationic activator. STH was successfully entrapped in the AOT/isooctane reverse micelles as well as GLD and was revealed to be capable of catalyzing the stoichiometric hydrogen transfer reaction between NADP+ and NADPH in reverse micelles. These results indicate that GLD and STH have potential for use in redox cofactor recycling in reverse micelles, which allows the use of catalytic quantities of NAD(P)H in organic media.     

Differing features of proteins in membranes may result in antioxidant or prooxidant action: opposite effects on lipid peroxidation of alcohol dehydrogenase and albumin in liposomal systems

SUMMARY

The influence of 3 thiol-containing compounds, bovine serum albumin (fatty acid free: BSA), glutathione (GSH) and yeast alcohol dehydrogenase (YADH) on lipid peroxidation in multilamellar liposomes, prepared from ox-brain phospholipid, was investigated. Thiol-compounds were added either before liposome formation, or after liposome formation; and their effects compared to a positive control. Bovine serum albumin (BSA), an acidic hydrophilic protein, displays a small, concentration dependent, antioxidant effect when added to preformed liposomes. A much larger antioxidant effect was observed when the BSA was entrapped inside the liposome, by adding BSA just prior to liposome preparation. In contrast, a Zn²⁺ containing redox enzyme, YADH, a basic hydrophobic membrane-associating protein, displays a large pro-oxidant effect at much lower concentrations especially when entrapped inside the liposome. This was observed also with GSH; but per mole of -SH, YADH was about 18 times as powerful a pro-oxidant perhaps because of structural changes to the membrane. Oxidized glutathione and N-acetylcysteine were also pro-oxidant (cysteine and cystine showed little effect). Formation of thiyl radicals may occur in the presence of iron ions with these pro-oxidant sulphur-containing compounds. Partial protection against lipid peroxidation was observed with EDTA, desferrioxamine and protoporphyrin (IX), potent iron-chelating agents.     

Measurements of hydrodynamic diameter of AOT reverse micelles containing lipase in supercritical ethane and its enzymatic reaction

The enzymatic reaction by aerosol-OT (AOT)reverse micelles containing lipase in supercritical ethane was examined and is the focus of this paper. The reverse micelles were formed under various conditions at which their hydrodynamic diameters were measured by using the dynamic light scattering spectrophotometer. The reverse micelles in supercritical ethane were formed in the range of Wo (water/surfactant) less than six. The hydrodynamic diameter of the reverse micelles ranged from 2 to 5 microm. The hydrolysis reaction of triolein by the lipase in reverse micelles was also examined. The observations indicate that lipase in AOT reverse micelles in supercritical ethane showed activity. The conversion of triolein increased with the increase in size of reverse micelles and Wo, and reached its maximum near the critical temperature. Moreover, although the conversion of triolein increased with pressure, it was independent of pressure near the critical temperature.     

Influence of Water on the Primary Photosynthetic Activity of Rhodospirillum Rubrum in Reverse Micelles

The effect of water on the primary photosynthetic activity of purple bacterium Rhodospirillum rubrum was studied in Hexadecane-Tween-Spane (HTS)- and phospholipid (PLC)-reverse micelles. Reverse micelles offer the possibility of modulating the amount of water to which enzymes and multienzymatic complexes are exposed. Fast bacteriochlorophyll (BChl) fluorescence induction kinetics and reaction centre absorption changes at 820 nm were used as an assay for the functional transfer of bacterial cells into HTS-reverse micelles and bacterial photosynthetic complexes (BPC) into PLC-reverse micelles. Both the bacterial cells and BPC showed an increase in the rate of primary photosynthetic activity by increasing the concentration of water in the reverse micelles. The bacterial cells could be kept viable for many hours in HTS-reverse micelles in presence of 6% (v/v) water. NMR studies indicated that the photosynthetic activity was affected by the availability of water in reverse micelles. The bacterial cells in HTS or BPC in PLC reverse micelles could be used to further understand the influence of water on the organisation and function of photosynthetic complexes. This revised version was published online in August 2006 with corrections to the Cover Date.     

Silica nanotubes-doped alginate gel for yeast alcohol dehydrogenase immobilization

A novel alginate–silica nanotubes (ALG–SiNTs) composite was prepared through the incorporation of silica nanotubes (SiNTs) into the alginate (ALG) gel followed by Ca²⁺ cross-linking for encapsulating yeast alcohol dehydrogenase (YADH, EC 1.1.1.1) from Saccharomyces cerevisiae. Pre-adsorption of YADH onto the surface of SiNTs before encapsulating in alginate gel was adopted to circumvent the enzyme leakage. AFM and SEM characterization confirmed that YADH molecules were substantially adsorbed on the SiNTs. SEM and EDX studies showed that the SiNTs homogenously distributed in alginate matrix. The enzyme leakage from ALG–SiNTs–YADH composite was remarkably reduced about 50% compared to that of ALG–YADH composite. Meanwhile, the optimum reaction condition, catalytic activity and kinetic parameters of immobilized YADH in ALG–SiNTs composite were studied. The results showed that stronger affinity between substrates and enzyme, higher activity retention, improved storage and operational stability were achieved when YADH was immobilized in ALG–SiNTs composite instead of ALG–YADH composite.     

Lipase-Catalyzed Reactions in Reverse Micelles Formed by Soybean Lecithin

Reverse micelles formed by soybean lecithin in isooctane were used as a reaction medium for both the lipase-catalyzed hydrolysis as well as the synthesis of lipids. Neither reaction appears to follow Michaelis-Menten kinetics and it is suggested that the rates are diffusion controlled. The hydrolysis of para-nitrophenylpalmitate (PNPP) and, in particular, the pH-dependency of the lipase-catalyzed hydrolysis was then examined. The highest rate of reaction occurred at pH_opt = 5–5.5, which was the same in water and lecithin reverse micelles, as well as in reverse micelles formed by bis(2-ethylhexyl)-sulfosuccinate (AOT) in isooctane. The dependence of the reaction rate on the water content of the micellar system was investigated for the same reaction. The maximal rate was found at an extremely low water content, i.e. at W_o = 2.2 (W_o = [H₂O]/[Lecithin]). The temperature stability of the lipase in lecithin reverse micelles was also studied and found to be greater than in aqueous solutions. Studies of the dependence of the relative initial velocity on temperature have shown that the highest rate in reverse micelles is obtained at 60d`C.     

Silica nanotubes-doped alginate gel for yeast alcohol dehydrogenase immobilization

A novel alginate–silica nanotubes (ALG–SiNTs) composite was prepared through the incorporation of silica nanotubes (SiNTs) into the alginate (ALG) gel followed by Ca²⁺ cross-linking for encapsulating yeast alcohol dehydrogenase (YADH, EC 1.1.1.1) from Saccharomyces cerevisiae. Pre-adsorption of YADH onto the surface of SiNTs before encapsulating in alginate gel was adopted to circumvent the enzyme leakage. AFM and SEM characterization confirmed that YADH molecules were substantially adsorbed on the SiNTs. SEM and EDX studies showed that the SiNTs homogenously distributed in alginate matrix. The enzyme leakage from ALG–SiNTs–YADH composite was remarkably reduced about 50% compared to that of ALG–YADH composite. Meanwhile, the optimum reaction condition, catalytic activity and kinetic parameters of immobilized YADH in ALG–SiNTs composite were studied. The results showed that stronger affinity between substrates and enzyme, higher activity retention, improved storage and operational stability were achieved when YADH was immobilized in ALG–SiNTs composite instead of ALG–YADH composite.     

Variation of transition-state structure as a function of the nucleotide in reactions catalyzed by dehydrogenases. 1. Liver alcohol dehydrogenase with benzyl alcohol and yeast aldehyde dehydrogenase with benzaldehyde

Primary intrinsic deuterium and 13C isotope effects have been determined for liver (LADH) and yeast (YADH) alcohol dehydrogenases with benzyl alcohol as substrate and for yeast aldehyde dehydrogenase (ALDH) with benzaldehyde as substrate. These values have also been determined for LADH as a function of changing nucleotide substrate. As the redox potential of the nucleotide changes from -0.320 V with NAD to -0.258 V with acetylpyridine-NAD, the product of primary and secondary deuterium isotope effects rises from 4 toward 6.5, while the primary 13C isotope effect drops from 1.025 to 1.012, suggesting a trend from a late transition state with NAD to one that is more symmetrical. The values of Dk (again the product of primary and secondary isotope effects) and 13k for YADH with NAD are 7 and 1.023, suggesting for this very slow reaction a more stretched, and thus symmetrical, transition state. With ALDH and NAD, the primary 13C isotope effect on the hydride transfer step lies in the range 1.3-1.6%, and the alpha-secondary deuterium isotope effect on the same step is at least 1.22, but 13C isotope effects on formation of the thiohemiacetal intermediate and on the addition of water to the thio ester intermediate are less than 1%. On the basis of the relatively large 13C isotope effects, we conclude that carbon motion is involved in the hydride transfer steps of dehydrogenase reactions.     

Mathematical modelling of the dehydrogenase catalyzed hexanol oxidation with coenzyme regeneration by NADH oxidase

The hexanol oxidation catalyzed by alcohol dehydrogenase from baker's yeast (YADH) has been investigated with two different forms of the biocatalyst: the isolated YADH as well as the YADH in the permeabilized whole cells. It was found that in this reaction, equilibrium is shifted to the reduction side. Hence, to increase the conversion it was necessary to regenerate NAD⁺. For that purpose, enzyme NADH oxidase isolated from Lactobacillus brevis was used. All biocatalysts were kinetically characterized. The overall reaction rate was described by the mathematical model which consisted of kinetics and balance equations. Due to the deactivation of NADH oxidase, only 50–58% hexanol was converted to hexanal in the batch reactor where the hexanol oxidation was catalyzed by isolated YADH. In the case of permeabilized baker's yeast cells, no enzyme deactivation occurred and 100% hexanol conversion in the hexanoic acid was detected.     

Strategies for enzymatic esterification in organic solvents: comparison of microaqueous, biphasic, and micellar systems.

The kinetics of butyl butyrate synthesis by a lipase from Mucor miehei in different types of organic media were investigated. The three systems studied were a microaqueous medium containing enzyme in suspension in hexane, a water-hexane two-phase system, and reverse micelles. The synthesis of butyl butyrate was possible in all cases because of a favorable partition of the ester into the organic solvent. A sufficient stirring rate was necessary to achieve good reaction rates in the case of the liquid-liquid biphasic medium. The effect of water content was different according to the type of system used. The dependence of reaction rate and of conversion yield on enzyme and substrate concentrations was also investigated. From an applied point of view, the best performances were obtained with either microaqueous or liquid-liquid two-phase systems. The use of reverse micelles can be advocated only in particular conditions, such as low enzyme concentration, compatible with the specific constraints it involves.     

Strictinin as an efficient antioxidant in lipid peroxidation

The antioxidant effect of strictinin (SOH), which was extracted from green tea leaves, against the peroxidation of linoleic acid in sodium dodecyl sulfate (SDS) and cetyl trimethylammonium (CTAB) micelles, against the peroxidation of low-density lipoprotein (LDL) and against oxidative hemolysis of human red blood cells (RBCs), has been studied. The peroxidation of linoleic acid and LDL, and oxidative hemolysis of RBCs were initiated thermally by a water-soluble azo initiator 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH), and the reaction kinetics in micelles and LDL were monitored by uptake of oxygen. The synergistic antioxidant effect of SOH with alpha-tocopherol (Vitamin E) was also studied by following the decay kinetics of alpha-tocopherol. Kinetic analysis of the antioxidation process demonstrates that SOH, used either alone or in combination with alpha-tocopherol, is an effective antioxidant against lipid peroxidation, but its effects significantly depend on the reaction medium.     

FTIR study of horseradish peroxidase in reverse micelles

Fourier transform infrared (FTIR) method was used to study the secondary structures of horseradish peroxidase (HRP) in aqueous solution and in reverse micelles for the first time. Results indicated that the structure of HRP in sodium bis(2-ethylhexy)sulfosuccinate (AOT) reverse micelles was close to that in aqueous solution. In cetyltrimethylammonium bromide (CTAB) and sodium dodecylfate (SDS) reverse micelles the position of some bands changed. Results indicated that the secondary structure had a close relationship with the surfactant species of the reverse micelles. Among the three types of reverse micelles, the system of AOT reverse micelles was probably the most beneficial reaction media to HRP.     

纳米磁性壳聚糖微球固定化酵母醇脱氢酶的研究

建立了以纳米级磁性壳聚糖微球(magnetic chitosan microspheres , M-CS)为载体固定化酵母醇脱氢酶(yeast alcohol dehydrogenase,YADH)的方法,优化了YADH的固定化条件,考察了固定化酶的性质。结果表明,M-CS 呈规则的圆球形,粒径在30nm 左右,具有较好的磁响应性。酵母醇脱氢酶固定化适宜条件为:50 mg 磁性壳聚糖微球,加入20mL 0.25 mg/mL 酵母醇脱氢酶(蛋白质含量)磷酸盐缓冲液(0.05 mol/L ,pH 7.0) ,在4 ℃固定2h。M-CS 容易吸附酵母醇脱氢酶,但吸附的酶量受载体与酶的比例、溶液的离子浓度、溶液pH的影响明显,而温度对吸附的酶量的影响则相对较弱。相对于游离的酵母醇脱氢酶,固定化酶的最适温度略有升高,可明显改善其热稳定性、酸碱稳定性、操作稳定性和贮存稳定性。     

Activity and stability of horse-liver alcohol dehydrogenase in sodium dioctylsulfosuccinate/cyclohexane reverse micelles

Horse liver alcohol dehydrogenase (EC 1.1.1.1) solubilized in sodium dioctylsulfosuccinate (AOT)/cyclohexane reverse micelles was used for the oxidation of ethanol and reduction of cyclohexanone in a coupled substrate/coenzyme recycling system. The activity of the enzyme was studied as a function of pH and water content. The enzyme was optimally active in microemulsions prepared with buffer of pH around 8. An increase in enzymatic activity was observed as a function of increasing water content. The Km values for the substrates were calculated based on the total reaction volume. The apparent Km for ethanol in reverse micelles was about eight times lower as compared to that in buffer solution, whereas the Km for cyclohexanone was almost unaltered. Storage and operational stability were investigated. It was found that the specific activity of the alcohol dehydrogenase operating in reverse micellar solution was good for at least two weeks. The steroid eticholan-3 beta-ol-17-one was also used as a substrate. In this case the reaction rate was approximately five times higher in a reverse micellar solution than in buffer.     

Liposomal encapsulation of yeast alcohol dehydrogenase with cofactor for stabilization of the enzyme structure and activity

Yeast alcohol dehydrogenase (YADH) with its cofactor nicotinamide adenine dinucleotide (NAD+) could be stably encapsulated in liposomes composed of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine). The YADH- and NAD+-containing liposomes (YADH-NADL) were 100 nm in mean diameter. The liposomal YADH and NAD+ concentrations were 2.3 mg/mL and 3.9 mM, respectively. A synergistic effect of the liposomal encapsulation and the presence of NAD+ was examined on the thermal stability of YADH at 45 and 50 degrees C. The enzyme stability of the YADH-NADL was compared to the stabilities of the liposomal YADH (YADHL) containing 3.3 mg/mL YADH without NAD+ as well as the free YADH with and without NAD+. Free YADH was increasingly deactivated during its incubation at 45 degrees C for 2 h with decrease of the enzyme concentration from 3.3 to 0.01 mg/mL because of the dissociation of tetrameric YADH into its subunits. At that temperature, the coexistence of free NAD+ at 3.9 mM improved the stability of free YADH at 2.3 mg/mL through forming their thermostable complex, although the stabilization effect of NAD+ was lowered at 50 degrees C. The turbidity measurements for the above free YADH solution with and without NAD+ revealed that the change in the enzyme tertiary structure was much more pronounced at 50 degrees C than at 45 degrees C even in the presence of NAD+. This suggests that YADH was readily deactivated in free solution due to a decrease in the inherent affinity of YADH with NAD+. On the other hand, both liposomal enzyme systems, YADH-NADL and YADHL, showed stabilities at both 45 and 50 degrees C much higher than those of the above free enzyme systems, YADH/NAD+ and YADH. These results imply that the liposome membranes stabilized the enzyme tertiary and thus quaternary structures. Furthermore, the enzyme activity of the YADH-NADL showed a stability higher than that of the YADHL with a more remarkable effect of NAD+ at 50 degrees C than at 45 degrees C. This was considered to be because even at 50 degrees C the stabilization effect of lipid membranes on the tertiary and quaternary structures of the liposomal YADH allowed the enzyme to form its thermostable complex with NAD+ in liposomes.     

Photodynamic effect in lysozyme: a kinetic study in different micellar media.

The influence of medium heterogeneity on the kinetics of the photodynamic effect on native protein lysozyme (Lyso), as well as the interaction of protein and the medium, anionic (SDS) micelles, neutral (Triton X-100) micelles and reversed micelles of AOT, were investigated at pH 8. The interaction between Lyso, Triton X-100 and SDS micelles was quantified by determining the respective associations constant (K(Lyso)). Values were 37 M(-1) for Triton X-100 and 514 M(-1) for SDS, indicating that the Lyso molecule binds Triton X-100 micelles effectively and SDS micelles even more strongly. Time-resolved phosphorescence detection (TRPD) indicates that the protein interacts with O2 (1deltag), with overall rate constants of the order of 10(8) M(-1)/S in direct micelles and 10(7) M(-1)/S in reverse micelles. Apparent reactive rate constants for eosin-sensitized photo-oxidation (singlet molecular oxygen [O2 (1deltag)]-mediated) of the protein were determined through oxygen uptake experiments for the direct micelles, while the fade in the protein fluorescence spectrum upon sensitized irradiation was used in AOT. The results indicate that the O2 (1deltag) attack on the interior of Lyso on amino acid residues, was more effective in leading to a photo-oxidative reaction in SDS and in Triton X-100 at surfactant concentrations < 1 x 10(-2) M than in a homogeneous solution. However, Lyso reactivity reached a maximum when the concentration of micelles was approximately 1 x 10(-5), the same as the protein concentration In AOT reverse micelles, the quenching rate constants decreased > 75% with respect to water. This effect can be attributed to the decrease in accessibility of the amino acid residues to O2 (1deltag).     

Cutinase-AOT interactions in reverse micelles: the effect of 1-hexanol

Cutinase encapsulated in dioctyl sulfosuccinate reverse micelles displays very low stability, undergoing fast denaturation due to an anchoring at the micellar interface. The denaturation process and the structure of the reverse micelle were characterized using biophysical techniques. The kinetics of denaturation observed from fluorescence match the increase of the hydrodynamic radius of reverse micelles. Denaturation in reverse micelles is mainly the unfolding of the three-dimensional structure since the decrease in the circular dichroism ellipticity in the far-UV range is very small. The process is accompanied by an increase in the steady-state anisotropy, as opposed to what happens for denaturation in aqueous solution.Since 1-hexanol used as co-surfactant in dioctyl sulfosuccinate reverse micelles slows or even prevents cutinase denaturation, its effect on cutinase conformation and on the size of reverse micelles was analyzed. When 1-hexanol is present, cutinase is encapsulated in a large reverse micelle, as deduced from dynamic light scattering. The large reverse micelle filled with cutinase was built from the fusion of reverse micelles according to a pseudo-unimolecular process ranging in time from a few minutes to 2h depending on the reverse micellar concentration. This slow equilibrium driven by the encapsulated cutinase has not been reported previously. The encapsulation of cutinase in dioctyl sulfosuccinate reverse micelles establishes a completely new equilibrium characterized by a bimodal population of empty and filled reverse micelles, whose characteristics depend greatly on the interfacial characteristics, that is, on the absence or presence of 1-hexanol.     

Deactivation and conformational changes of cutinase in reverse micelles

Deactivation data and fluorescence intensity changes were used to probe functional and structural stability of cutinase in reverse micelles. A fast deactivation of cutinase in anionic (AOT) reverse micelles occurs due to a reversible denaturation process. The deactivation and denaturation of cutinase is slower in small cationic (CTAB/1-hexanol) reverse micelles and does not occur when the size of the cationic reverse micellar water-pool is larger than cutinase. In both systems, activity loss and denaturation are coupled processes showing the same trend with time. Denaturation is probably caused by the interaction between the enzyme and the surfactant interface of the reversed micelle. When the size of the empty reversed micelle water-pool is smaller than cutinase (at W0 5, with W0 being the water:surfactant concentration ratio) a three-state model describes denaturation and deactivation with an intermediate conformational state existing on the path from native to denaturated cutinase. This intermediate was clearly detected by an increase in activity and shows only minor conformational changes relative to the native state. At W0 20, the size of the empty water-pool was larger than cutinase and the data was well described by a two-state model for both anionic and cationic reverse micelles. For AOT reverse micelles at W0 20, the intermediate state became a transient state and the deactivation and denaturation were described by a two-state model in which only native and denaturated cutinase were present. For CTAB/1-hexanol reverse micelles at W0 20, the native cutinase was in equilibrium with an intermediate state, which did not suffer denaturation. 1-Hexanol showed a stabilizing effect on cutinase in reverse micelles, contributing to the higher stabilities observed in the cationic CTAB/1-hexanol reverse micelles. Copyright 1998 John Wiley & Sons, Inc.