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.     

Glucosaminephosphate synthase of human liver.

Although human liver contains glucosaminephosphate synthase (glucosaminephosphate isomerase (glutamine-forming), EC 5.3.1.19), its activity is rapidly lost during the course of extraction. The inactivation, however, is largely prevented if the extraction medium contains isopropanol at 1% concentration; using these "stabilized" extracts, the glucosaminephosphate synthase activity of human liver has been shown to be similar to the activity previously reported in rat liver. The enzyme precipitated from these extracts by (NH4)2SO4 is inhibited by UDP-N-acetylglucosamine, the concentration required to produce a half-maximal inhibition being 6 muM. These results seem to be sufficient to postulate that glucosaminephosphate synthase is important for UDP-N-acetylglucosamine synthesis in human liver. In contrast to the rat liver enzyme, the (NH4)2SO4-precipitated human liver enzyme is resistant to trypsin and undergoes no conversion reaction when incubated with glucose 6-phosphate.     

Refolding of denatured lysozyme by water-in-oil microemulsions of sucrose fatty acid esters

Water-in-oil (w/o) microemulsion of sucrose fatty acid ester was used to renature denatured hen egg white lysozyme without aggregation. After lysozyme was denatured in 5 M guanidine hydrochloride for 24 h, the resultant denatured lysozyme was held in the microemulsion, overnight at 25°C. Renatured lysozyme was transferred from the microemulsion phase to the recovery aqueous phase by conventional liquid-liquid extraction. The enzymatic activity of the recovered lysozyme was 93%.     

Stability and activity of 20 beta-hydroxysteroid dehydrogenase in microemulsion of non-ionic detergents

We report here the formation of a microemulsion with non-ionic detergents and cyclohexane. The activity and stability of 20 beta-hydroxysteroid dehydrogenase solubilized in all water systems and in microemulsions of Nonidet P-40: Triton X-35/water/cyclohexane was investigated. In the microemulsion the activity depended on the molecular ratio of water to surfactant (Wo); maximal activity was obtained at Wo of 8.4. The stability in the microemulsion was higher at Wo = 11.75 i.e. the enzyme, retained about 50% of activity after eight days.     

Three Systems Used for Biocatalysis in Organic Solvents a Comparative Study

The activity and operational stability of horse liver alcohol dehydrogenase (HLADH) and α-chymotrypsin were investigated in three systems commonly used for biocatalysis in organic solvents:

1. enzyme adsorbed on a solid support (celite) and added to the organic solvent (isooctane)

2. enzyme powder directly added to the organic solvent (isooctane).

3. enzyme dissolved in a microemulsion (AOT/isooctane).

The activity and the operational stability in all systems were strongly dependent on the water content. The initial reaction rate was high in both the microemulsion and the celite system, but was much lower when adding the enzymes directly to the organic solvent. HLADH was observed to be more stable when added directly to the organic solvent or dissolved in the microemulsion than when adsorbed on celite, whereas for α-chymotrypsin stability was higher when adsorbed on celite or added directly to the organic solvent. For a hydrolytic reaction, a microemulsion was preferred due to the high water content. When adding the enzymes directly to the organic solvent both HLADH and chymotrypsin were adsorbed strongly to the glass walls of the reaction vessel. None of the systems were superior in all respects for the two enzymes studied.     

Evaluation of steady-state kinetic parameters for enzymes solubilized in water-in-oil microemulsion systems.

1. Equations are derived for the steady-state kinetics of substrate conversion by enzymes confined within the water-droplets of water-in-oil microemulsion systems. 2. Water-soluble substrates initially confined within droplets that do not contain enzyme are assumed to be converted into product only after they enter enzyme-containing droplets via the inter-droplet exchange process. 3. Hyperbolic (Michaelis-Menten) kinetics are predicted when the substrate concentration is varied in microemulsions of fixed composition. Both kcat. and Km are predicted to be dependent on the size and concentration of the water-droplets in the microemulsion. 4. The predicted behaviour is shown to be supported by published experimental data. A physical interpretation of the form of the rate equation is presented. 5. The rate equation for an oil-soluble substrate was derived assuming a pseudo-two-phase (oil & water) model for the microemulsion. Both kcat. and Km are shown to be independent of phi aq. Km is larger than the aqueous solution value by a factor approximately equal to the oil/water partition coefficient of the substrate. The validity of the rate equation is confirmed by published data.     

Novel reactive perstraction system applied to the hydrolysis of penicillin G

The activity of penicillin acylase has been studied in aqueous and organic solvents, as free enzyme as well as immobilized within the membrane of liquid-core capsules. The activity of the enzyme is inhibited by the accumulation of the products of the hydrolysis reaction, namely phenyl acetic acid (PAA). In order to overcome this inhibition a range of organic solvents were tested for use in in situ product recovery. Of these solvents dibutyl sebacate (DBS) was chosen due to the rapid extraction rate, the high logP and to facilitate capsule production. The extraction efficiency at pH 3.5 for PAA was >80% for phase ratios of >50% free solvent with partition coefficients of 8 and 0.7 for PAA and penicillin G (PenG), respectively, thereby showing that PAA could be selectively extracted at pH 3.5 and 25 degrees C. Liquid-core capsules containing DBS were shown to efficiently remove PAA selectively and the PAA could be effectively back-extracted and the capsules re-used in a three-stage process resulting in high product separation. Immobilization of penicillin acylase onto the capsule membranes resulted in increased operational stability of the enzyme and a very high enzyme activity. Over 53.3% of the PAA formed could be recovered in the capsule core with a concentration over sevenfold higher than in the aqueous phase. Higher extraction efficiencies could be obtained by varying the substrate concentration and number of capsules. The enzyme immobilized on capsules could be stored for over 4 months at pH 8 and 4 degrees C with no loss of activity. Over 80% of the initial activity could be recovered over five repeated batch cycles of the bioconversion process. The importance of capsular perstraction and reactive capsular perstraction has been clearly demonstrated.     

Enzyme-Catalyzed oxidation of cholesterol in physically characterized water-in-oil microemulsions

The enzymatic conversion of cholesterol to cholestenone by cholesterol oxidase (Brevibacterium sp.)in reversed micelles in a system composed of AOT/isooctane/water/cholesterol has been examined. The catalytic activity of the enzyme was correlated with the physicochemical properties of water in water-in-oil (w/o) microemulsion systems. In a system consisting of 3 wt % AOT in isooctane, reversed micelles started to form as the [H(2)O]/[AOT] (e.g., the w(0)) ratio increased above 4-5. The formation of reversed micelles with a core of neat (bulk) water was verified from determinations of both the partial molar volume of water and the scissors vibration of water [with Fourier transform infrared (FTIR) spectroscopy] in the w/o microemulsion systems. A plot of enzyme activity vs. w(0) indicated that the hydration of enzyme molecules per se was not sufficient to give rise to catalytic activity. Instead, it appeared that the formation of an aqueous micellar core was necessary for full activation of the enzyme. Based on micelle size distribution analysis, it was estimated that about one micelle per one thousand contained an enzyme molecule. Since the apparent reaction rate could be markedly enhanced by increasing the enzyme/water ratio, we conclude that the number of enzyme-containing micelles was an important rate-limiting factor in the system.     

The DFPase from Loligo vulgaris in sugar surfactant-based bicontinuous microemulsions: structure, dynamics, and enzyme activity

The enzyme diisopropyl fluorophosphatase (DFPase) from the squid Loligo vulgaris is of great interest because of its ability to catalyze the hydrolysis of highly toxic organophosphates. In this work, the enzyme structure in solution (native state) was studied by use of different scattering methods. The results are compared with those from hydrodynamic model calculations based on the DFPase crystal structure. Bicontinuous microemulsions made of sugar surfactants are discussed as host systems for the DFPase. The microemulsion remains stable in the presence of the enzyme, which is shown by means of scattering experiments. Moreover, activity assays reveal that the DFPase still has high activity in this complex reaction medium. To complement the scattering experiments cryo-SEM was also employed to study the microemulsion structure.     

A HPLC method for the analysis of germacrone in rabbit plasma and its application to a pharmacokinetic study of germacrone after administration of zedoary turmeric oil

A validated new, simple and highly sensitive reversed-phase HPLC method is developed for studying the pharmacokinetics of germacrone after intravenous administration of zedoary turmeric oil (ZTO) oil-in-water microemulsion. The method did not require a complex and expensive equipment. A high extraction recovery (>80%) of germacrone was obtained. Linear calibration curves obtained with the peak-area ratio (y) of germacrone to internal standard (tanshinoneIIA) versus drug concentration (x) were found to be linear between 8.08 and 808 ng/ml. The limit of quantitation was 8.08 ng/ml.The monitored compounds were completely separated from others in ZTO and from endogenous species in plasma by HPLC. Pharmacokinetic investigations were performed on 18 male rabbits after intravenous administration of ZTO microemulsion via the ear vein at germacrone doses of 3.2, 6.4 and 9.6 mg/kg. The plasma concentration-time data fit to a two-compartment intravenous model with a weight of 1/C(2) (C: germacrone concentration in plasma). Germacrone exhibited linear pharmacokinetics after intravenous administration of ZTO microemulsion to rabbits over the germacrone dose range 3.2-9.6 mg/ml.     

Extraction of collagenase from the 6000 times g sediment of uterine and skin tissues of mice. A comparative study.

An enzyme capable of digesting native collagen in solution at neutral pH was extracted from the 6 000 times g sediment of the involuting uterus of the mouse and of the back skins of mice and rats. The collagenase could be dissociated at cold-room temperature from the sediment in about equal amounts when neutral Tris buffer containing 1.0M NaCl or 5M urea was used for the extraction step. The enzyme has been concentrated by ammonium sulfate precipitation and the activity was measured by using [14C]collagen in solution at pH 7.5. Collagen breakdown products were identified by disc electrophoresis. The amount of enzyme extracted was a function of temperature and salt concentration. As 5M urea extracted collagenase from the sediment in a relatively short time, this method of extraction seems to be a useful tool for serial experiments in the study of collagenase activity in collagen-rich tissues.     

反胶束萃取技术分离胰激肽原酶

研究了用十六烷基三甲基溴化铵（CTAB）/正己醇/正辛烷反胶束溶液萃取和反萃取商业用胰激肽原酶时,水相pH值、离子强度和种类、CTAB浓度和助表面活性剂浓度等因素对分离效率的影响,并从反胶束微观结构给予解释。结果表明：［CTAB］＝0.02 mol•L-1,正己醇／正辛烷(V/V)＝1：5,萃取pH＝9.0,反萃pH＝7.0,萃取［KBr］＝0.1 mol•L-1,反萃［KBr］＝1.5 mol•L-1,反萃取加15％乙醇(V/V)时,萃取率接近100%,反萃取活性回收得率在80%以上。商业用酶的纯化倍数最高为1.97倍,粗酶为7.15倍,且粗酶纯化后比活在200U／mg以上,电泳分析证实了纯化效果,显示了很好的工业前景。     

Beta-hydroxysteroid dehydrogenase: activity in microemulsion and extraction from Pseudomonas testosteroni cells with microemulsion

The stability of purified beta-hydroxysteroid dehydrogenase activity measured as a function of time was good in buffered cationic and non-ionic microemulsions. The use of 1-pentanol and 1-hexanol in place of 1-butanol as cosurfactant gave increased activity and stability. The NAD+ Michaelis constant was 0.22 mM in buffer and 3.5 mM in waterpool concentration in microemulsion. Proteins, among them beta-hydroxysteroid dehydrogenase, were extracted from Pseudomonas testosteroni with cationic microemulsion, thus indicating that microemulsions may be utilized in protein release from cells.     

Protein extraction by reverse micelles: a study of the factors affecting the forward and backward transfer of alpha-chymotrypsin and its activity

alpha-chymotrypsin is taken as a model protein to investigate three aspects of the protein extraction by reverse micelles: (1) the comparison between the two forward transfer techniques, i.e., the liquid-liquid and the solid state-liquid transfer; (2)the back-transfer, i.e., the capability of the protein to be recovered from the micellar solution; and (3) the maintainance of the enzyme activity at the end of the extraction cycle. Concerning the forward transfer from the liquid phase, we study first the effect of salt initially present in the aqueous phase on the equilibrium concentration of the extracted species; further, we study the forward protein extraction from the solid state, and the effect of pH, salt, and protein concentration on the transfer efficiency. Concerning the back transfer, we find the somewhat surprising result, that the percentage of protein back-extraction depends on the type and concentration of salt used for the forward transfer. Preliminary data concerning an alternative method for the back-transfer using silica gel to liberate the protein from the micellar environment, are presented. Finally, it is found that the enzyme activity depends again on the type and concentration of salt used for the forward transfer.     

Kinetics in microemulsion V. Glucose oxidase catalyzed oxidation of beta-D-glucose in aqueous, micellar and water-in-oil microemulsion media

The oxidation of beta-D-glucose by the enzyme glucose oxidase was studied in aqueous medium, in solutions of surfactants AOT (2-ethylhexylsulfosuccinate, sodium salt) TX-100 (polyethylene glycol p-tert octyl phenyl ether) and in w/o microemulsion medium (water/AOT/decane) at different water/AOT mole ratio (omega), pH, temperature and in presence of additives. The time-dependent activities of the enzyme in aqueous and microemulsion media were determined. The catalytic process was retarded in the presence of TX-100 and AOT. In microemulsion medium, kcat values exhibited a deformed W-shaped profile with omega. At pH 7, a maximum value of kcat was observed at omega = 10.6. The kcat values were found to be higher in microemulsion medium than in aqueous medium at both pH's 7 and 8. Activation parameters for the kinetic process were evaluated together with the thermodynamics of the enzyme-substrate Michaelis complex. The deltaG* was lower, whereas deltaH* and deltaS* were higher in microemulsion than in water. The Michaelis constant, KM was also lower in microemulsion. The inhibition effects of the additives, NaNO3 and NaC were studied in both aqueous and microemulsion media by examining their influences on catalytic constant, kcat and Michaelis constant KM. In microemulsion, both the additives NaNO3 and NaC produced non-competitive inhibition.     

Anti-yeast activity of a food-grade dilution-stable microemulsion

The anti-yeast activities of a food-grade dilution-stable microemulsion against Candida albicans and Saccharomyces cerevisiae have been studied. The weight ratio of the formulated microemulsion is glycerol monolaurate (GML)/propionic acid/Tween 80/sodium benzoate (SB)/water = 3:9:14:14:24. Results of anti-yeast activity on solid medium by agar diffusion method showed that the anti-yeast activity of the microemulsion at 4.8 mg/ml was comparable to that of natamycin at 0.1 mg/ml as positive control. Results of anti-yeast activity in liquid medium by broth dilution method showed that the growth of both C. albicans and S. cerevisiae was completely inhibited when the liquid medium containing 10⁶ cfu/ml was treated with 1.2 mg/ml microemulsion, which was determined as minimum fungicidal concentration. The kinetics of killing results showed that the microemulsion killed over 90% yeast cells rapidly within 15 min and caused a complete loss of viability in 120 min. Among the components, SB and GML had a similar anti-yeast activity, followed by propionic acid, while Tween 80 exhibited no activity and could not enhance the anti-yeast activities of these components, and it was revealed that the anti-yeast activity of the microemulsion was attributed to a combination of propionic acid, GML, and SB. The anti-yeast activity of the microemulsion was in good agreement with the leakage of 260-nm absorbing materials and the observation of transmission electron microscopy, indicating that the microemulsion induced the disruption and dysfunction of the cell membrane.     

Biodegradation of pesticides in nonionic water-in-oil microemulsions of tween 85: Relationship between micelle structure and activity

Water-in-oil microemulsion systems have been studied in recent years for a number of applications in protein separation and enzymology. Although it is well established that reversed micelle systems provide an excellent medium for nonaqueous biocatalytic studies, there is still much speculation as to the interaction of the enzyme with the surfactant interface. Polyoxyethylene sorbitan trioleate (Tween 85) is a nonionic surfactant which has some interesting properties for microemulsion formation and protein solubilization. In conjunction with a separate article describing the structural features of Tween 85 reversed micelles in hexane with isopropanol as a cosurfactant, this work describes the activity of an enzyme, organophosphorus hydrolase, for degrading organophosphorus pesticides in this microemulsion system. Ternary phase diagrams were constructed to outline the phase boundaries at different temperatures and isopropanol concentrations, which elucidate the role of the cosurfactant alcohol, as well as some features of micelle structure. Kinetic and stability studies with organophosphorus hydrolase show the effect of enzyme partitioning between the micelle surfactant layer and aqueous core. (c) 1994 John Wiley & Sons, Inc.     

Characterization of a calmodulin-stimulated adenylate cyclase from abalone spermatozoa

Abalone sperm adenylate cyclase activity is particulate in nature and displays a high Mg2+-supported activity (Mg2+/Mn2+ = 0.8) as compared to other sperm adenylate cyclases. Approximately 90% of the enzyme activity in crude homogenates is inhibited by EGTA in a concentration-dependent manner which is overcome by added micromolar free Ca2+. The EGTA-inhibited Ca2+-stimulated enzyme activity is also inhibited by phenothiazines. Added calmodulin, however, has no effect on enzyme activity prepared from crude homogenates. Preparation of a twice EGTA-extracted 48,000 X g pellet fraction yields a particulate enzyme activity that can be stimulated 10-65% by added calmodulin in the presence of micromolar free Ca2+. Detergent extraction (1% Lubrol PX) of the EGTA-washed 48,000 X g pellet solubilizes 2-5% of the total particulate adenylate cyclase activity, and this solubilized enzyme is activated up to 125% by calmodulin. The ability of the different enzyme preparations to be stimulated by calmodulin is inversely proportional to the endogenous calmodulin concentration. Calmodulin stimulation of the Lubrol PX-solubilized enzyme is specific to this Ca2+-binding protein and is mediated as an effect on the velocity of the enzyme. This stimulation is completely Ca2+ dependent and is fully reversible. These data suggest that the control of sperm cAMP synthesis by changes in Ca2+ conductance may be mediated via this Ca2+-binding protein.     

Production of β-galactosidase by Kluyveromyces marxianus MTCC 1388 using whey and effect of four different methods of enzyme extraction on β-galactosidase activity

Whey containing 4.4% (w/v) lactose was inoculated with Kluyveromyces marxianus MTCC 1389 for carrying out studies related to β-galactosidase production. β-galactosidase activity was found to be maximum after 30 h and further incubation resulted in decline in activity. The maximum cell biomass of 2.54 mg mL⁻¹ was observed after 36 h of incubation. Lactose concentration dropped drastically to 0.04 % from 4.40% after 36 h of incubation. Out of the four methods tested for extraction of enzyme, SDS — Chlorofom method was found to be best followed by Toluene — Acetone, sonication and homogenization with glass beads in that order. It could be concluded through this study that SDS — Chloroform is cheap and simple method for enzyme extraction from Kluyveromyces cells, which resulted in higher enzyme activity as compared to the activity observed using the remaining extraction methods. The study could also establish that whey could effectively be utilized for β-galactosidase production thus alleviating water pollution problems caused due to its disposal into the water streams.     

沙棘果渣总黄酮提取工艺优化及抗氧化活性研究

利用果胶酶协同超声波法,对沙棘果渣有效成分总黄酮的提取工艺及其抗氧化活性进行了研究。以提取率为指标,通过酶用量、液料比、乙醇浓度、提取时间、提取温度、超声功率等单因素分析,选定酶用量、液料比、超声提取时间3个因素进行响应面试验,确定提取优化工艺条件为:果胶酶用量5.1%,液料比41∶1,超声提取时间81 min,此条件下,沙棘果渣总黄酮提取率达到8.91 mg/g;以BHT为阳性对照,进行了抗氧化活性研究,得出沙棘果渣总黄酮提取液浓度为0.14 mg/mL时,对DPPH自由基的清除率达到了94.42%;提取液浓度为1.2 mg/mL时,对羟自由基和超氧阴离子的清除率分别为83.10%和43.41%。整体来看,沙棘果渣总黄酮具有较强的抗氧化能力。