Biocatalysis of tyrosinase using catechin as substrate in selected organic solvent media

The enzymatic activity of mushroom tyrosinase was investigated using catechin as substrate in selected organic solvent media. The results showed that optimal tyrosinase activity was obtained at pH 6.2, 6.6, 6.0 and 6.2 in the organic solvent media of heptane, toluene, dichloromethane, and dichloroethane, respectively, and at a temperature between 25°C and 27.5°C. In addition, the kinetic studies showed that the K_m values were 5.38, 1.03, 2.52 and 4.03 mM, for the tyrosinase-catechin biocatalysis in the reaction media of heptane, toluene, dichloromethane, and dichloroethane, respectively, while the corresponding V_max values were 1.22×10⁻³, 0.33×10⁻³, 1.47×10⁻³ and 1.20×10⁻³ δA per μg protein per second, respectively. The use of acetone as co-solvent for the tyrosinase-catechin biocatalysis showed that acetone concentrations ranging from 5% to 30% (v/v) in the heptane reaction medium produced a decrease of 4.3% to 96.7% in tyrosinase activity. The results also indicated that the presence of 12.5% acetone in the reaction medium of dichloromethane, and 22.0% in those of toluene and dichloroethane produced a maximal increase of 42.6%, 92.1% and 71.8%, respectively, in tyrosinase activity. However, the overall findings indicated that additional increases in acetone concentration resulted in an inhibition of tyrosinase activity.     

Biocatalysis of immobilized chlorophyllase in a ternary micellar system.

The immobilization of chlorophyllase was optimized by physical adsorption on various inorganic supports, including alumina, celite, Dowex-1-chloride, glass beads and silica gel. The enzyme was also immobilized in different media, including water, Tris-HCl buffer solution and a ternary micellar system containing Tris-HCl buffer solution, hexane and surfactant. The highest immobilization efficiency (84.56%) and specific activity (0.34 mumol hydrolyzed chlorophyll mg protein-1 per min) were obtained when chlorophyllase was suspended in Tris-HCl buffer solution and adsorbed onto silica gel. The effect of different ratios of chlorophyllase to the support and the optimum incubation time for the immobilization of chlorophyllase were determined to be 1-4 and 60 min, respectively. The experimental results showed that the optimum pH and temperature for the immobilized chlorophyllase were 8.0 and 35 degrees C, respectively. The use of optimized amounts of selected membrane lipids increased the specific activity of the immobilized chlorophyllase by approximately 50%. The enzyme kinetic studies indicated that the immobilized chlorophyllase showed a higher affinity towards chlorophyll than pheophytin as substrate.     

Whole cell yeast biotransformations in two-phase systems: Effect of solvent on product formation and cell structure

Summary Biotransformation of benzaldehyde and pyruvate to (R)-phenylacetyl carbinol bySaccharomyces cerevisiae was investigated in two-phase aqueous-organic reaction media. With hexane as organic solvent, maximum biotransformation activity was observed with a moisture content of 10%. Of the organic solvents tested, highest biotransformation activities were observed with hexane and hexadecane, and lowest activities occurred with chloroform and toluene. Biocatalyst samples from biphasic media containing hexane, decane and toluene manifested no apparent cell structural damage when examined using scanning electron microscopy. In contrast, cellular biocatalyst recovered from two-phase systems containing chloroform, butylacetate and ethylacetate exhibited damage in the form of cell puncturing after different incubation periods. Phospholipids were detected in reaction media from biocatalytic systems which exhibited cell damage in electron micrographs. Phospholipid release was much lower in the two-phase systems containing toluene or hexane or in 100% aqueous biocatalytic system.     

Enzymes in Organic Synthesis VII—Enzymatic Activity of Hrp After Chemical Modification of the Carbohydrate Moiety

The carbohydrate moiety of horseradish peroxidase was conjugated with hexadecylamine or octylamine in a micellar medium. Recovery and purification of these conjugates was facilitated by the short length of the added spacers. The modification increased the liposolubility of the enzyme without detracting from its catalytic activity. For the hexadecylamine conjugate, the optimum reaction temperature was increased by 10d`C. In addition, activity in organic solvents, such as toluene or chloroform, remained high, even at 70d`C.     

Antimicrobial activity of volatile component and various extracts ofEnteromorpha linza (Linnaeus) J. Agardh from the coast of Izmir, Turkey

The methanol, dichloromethane, hexane, chloroform and volatile components ofEnteromorpha linza were testedin vitro for their antimicrobial activity against five Gram-positive, four Gram-negative bacteria andCandida albicans ATCC 10239. GC-MS analysis of the volatile components ofE. linza resulted in the identification of 35 compounds which constituted 84.76% of the total compounds. The volatile components ofE. linza consisted of n-tetratriacontane (8.45%), 1-heptadecanamine (6.65%) and docosane (6.46%) as major components. The methanol and chloroform extracts showed more potent antimicrobial activity than hexane and dichloromethane extracts. The volatile oils of these algae did not remarkably inhibit the growth of tested microorganisms.     

Preparation of a biodegradable oil absorber and its biodegradation

The biodegradable oil absorption resin (B-PEHA) was prepared by suspension polymerization, and its preparation was confirmed by Fourier transform infrared analysis. The oil absorption capacities of the prepared B-PEHA were: chloroform 30.88, toluene 19.75, xylene, 18.78, THF 15.96, octane 11.43, hexane 9.5, diesel oil 12.80, and kerosene 13.79 g/g. The biodegradation of the prepared B-PEHA was also investigated by determination of reduced sugar produced after enzymatic hydrolysis, thermogravimetric analysis, and incubation with Aspergillus niger. The biodegradation of B-PEHA was ~18%.     

The effect of cultivation media and washing whole-cell biocatalysts on monoamine oxidase catalyzed oxidative desymmetrization of 3-azabicyclo[3,3,0]octane

It is well known that washing whole-cells containing enzyme activities after fermentation, but prior to biocatalysis can improve their activity in the subsequent reaction. In this paper, we quantify the impact of both the fermentation media and cell washing on the performance of whole-cell biocatalysis. The results are illustrated using a recombinant monoamine oxidase (expressed in Escherichia coli, used in resting state) for the oxidative desymmetrization of 3-azabicyclo[3,3,0]octane. It was shown that the need for washing biocatalyst prior to use in a reaction is dependent upon growth medium. Unlike cells grown in LB medium, washing of the cells was essential for cells grown on TB medium. With TB media, washing the cells improved the final conversion by approximately a factor of two. Additionally, over 50-fold improvement was achieved in initial activity. A potential reason for this improvement in activity was identified to be the increase in transfer of substrates across the cell membrane as a result of cell washing.     

Immobilization of lipoxygenase in an alginate-silicate solgel matrix: formation of fatty acid hydroperoxides

A method for the immobilization of lipoxygenase (LOX) in an alginate-silicate gel matrix was developed. In this method, a mixture of calcium alginate beads and LOX in borate buffer are dispersed into a hexane solution of tetramethoxy-ortho-silicate (TMOS). Hydrolysis of the TMOS gives products that permeate and co-polymerize with the alginate gel to form a colloid within the beads that entraps the LOX. Optimum reaction conditions for sol-gel entrapment of LOX are at pH 9.0 in 0.2M borate buffer. The composite gel, after isolation and vacuum drying, had excellent protein retention that has good enzyme activity and stability at room temperature. The activity of the entrapped LOX was less than the activity of the free enzyme. However, the activity of the immobilized LOX can be restored by the addition of borate buffer and glycerol, or borate buffer saturated with an organic solvent. In contrast to the free enzyme in solution, which loses its activity in less than one day, sol-gel entrapped LOX retains its activity at ambient temperature for at least 25 days and can be recycled. This report demonstrates that the sol-gel entrapment method for immobilizing LOX can be useful in developing a process for the oxidation of polyunsaturated fatty acids.     

Comparison of tyrosinase activity and stability in aqueous and nearly nonaqueous environments

The activity and stability of tyrosinase were compared in aqueous and two nearly nonaqueous environments (a low-water solvent system and reversed micelles). Initial rates of oxidation of methyl- and butyl-catechols in aerosol OT, sodium di-2-ethylhexylsulfosuccinate, (AOT)/isooctane micelles were higher than in aqueous solution, showing superactivity, whereas lower rates were obtained in cetyltri-methylammonium bromide (CTAB)/hexane/chloroform micelles and in chloroform containing celite-supported enzyme. The enzyme was most stable in chloroform, whereas half-lives in aqueous buffer and in both AOT and CTAB micelles were lower. The optimal reaction temperatures were higher in both micelles than in water but lower in chloroform. Thus, tyrosinase was active in ≤3.5% v/v water with apparent K_m, V_max, and activation energies reasonably similar to those in aqueous solution.     

On the application of the Clark oxygen electrode to the study of enzyme kinetics in apolar solvents: the catalase reaction.

A method for recording O2 concentrations in nonconducting organic media with the Clark oxygen electrode was developed. Spontaneous oxidation of Na2S2O4 and the enzymatic reduction of NaBO3 or H2O2 by bovine liver catalase trapped in hydrated micelles of dioctylsulfosuccinate (AOT)/toluene were used as model systems. O2 titration with the above systems showed that air-saturated 1.6 M H2O/0.2 M AOT/toluene media contain seven times more O2 (1.4 mM) than aqueous solutions (0.2 mM). The measured Km values of catalase for NaBO3 and H2O2 in organic media were Kmov = 15 and 17 mM, respectively, whereas in aqueous buffer the values were 45 and 54 mM. In the toluene media, catalase activity increased with the W0 (H2O/AOT molar ratio) of the micellar preparation, reaching maximal activity at W0 = 10-12; under this condition, the catalytic center activity (Kp) of H2O2 was 7 x 10(6) min-1, similar to that obtained in the aqueous buffer (H2O2 = 7 x 10(6) min-1). It was found that the optimal pH for catalase in toluene media (pH 8.0) was shifted 1.0 unit compared to that in the aqueous buffer (pH 7.0). On the other hand, catalase was severely inhibited by NaN3 in both media. Thus, polarography based on the Clark oxygen electrode seems to be an easy, rapid, and sensitive technique for studying enzyme reactions consuming or evolving O2 in apolar media.     

含离子液体体系中固定化细胞转化甘草酸生成单葡萄糖醛酸基甘草次酸

研究疏水性离子液体1-丁基-3-甲基咪唑六氟磷酸盐([BMIM]PF6)与醋酸-醋酸钠缓冲液两相体系中,固定化产紫青霉Penicillium purpurogenum Li-3细胞转化甘草酸(GL)生成单葡萄糖醛酸基甘草次酸(GAMG)的反应,并与缓冲液单相体系作为对照.确定了在[BMIM]PF6/缓冲液两相体系中,最适离子液体加入比例、缓冲液pH、反应温度、底物浓度分别为10%、5.8、35℃和6.0mmol/L,在此条件下反应58h,甘草酸转化率为87.03%,比缓冲液单相体系提高了15.02%.离子液体循环使用8次后,回收利用率维持在85.28%.主产物GAMG和副产物甘草次酸(GA)在两相体系中得到有效分离,为后续产物分离带来便利.     

Alterations to the catalytic properties of polyphenoloxidase in detergentless microemulsions and ternary water-organic solvent mixtures

Summary The catalytic properties of mushroom polyphenoloxidase could be substantially altered by entrapment into hexane- and toluene-based microemulsions stabilized with isopropanol. The fast irreversible inactivation and drastic substrate inhibition of the enzyme were significantly reduced in detergentless microemulsions in comparison to conventional aqueous media. Similar changes in the catalytic behavior of polyphenoloxidase were observed in the normal ternary solutions of hexane-(toluene)-isopropanol-water, and in the H-bonded aggregates of isopropanol and water in toluene, but not in hexane.     

Laccase-catalyzed oxidation of phenolic compounds in organic media

Rhus vernificera laccase-catalyzed oxidation of phenolic compounds, i.e., (+)-catechin, (−)-epicatechin and catechol, was carried out in selected organic solvents to search for the favorable reaction medium. The investigation on reaction parameters showed that optimal laccase activity was obtained in hexane at 30 °C, pH 7.75 for the oxidation of (+)-catechin as well as for (−)-epicatechin, and in toluene at 35 °C, pH 7.25 for the oxidation of catechol. E_a and Q₁₀ values of the biocatalysis in the reaction media of the larger log p solvents like isooctane and hexane were relatively higher than those in the reaction media of lower log p solvents like toluene and dichloromethane. Maximum laccase activity in the organic media was found with 6.5% of buffer as co-solvent. A wider range of 0–28 μg protein/ml in hexane than that of 0–16.7 μg protein/ml in aqueous medium was observed for the linear increasing conversion of (+)-catechin. The kinetic studies revealed that in the presence of isooctane, hexane, toluene and dichloromethane, the K_m values were 0.77, 0.97, 0.53 and 2.9 mmol/L for the substrate of (+)-catechin; 0.43, 0.34, 0.14 and 3.4 mmol/L for (−)-epicatechin; 2.9, 1.8, 0.61 and 1.1 mmol/L for catechol, respectively, while the corresponding V_max values were 2.1 × 10⁻², 2.3 × 10⁻², 0.65 × 10⁻² and 0.71 × 10⁻² δA/μg protein min); 1.8 × 10⁻², 0.88 × 10⁻², 0.19 × 10⁻² and 1.0 × 10⁻² δA/μg protein min); 0.48 × 10⁻², 0.59 × 10⁻², 0.67 × 10⁻² and 0.54 × 10⁻² δA/μg protein min), respectively. FT-IR indicated the formation of probable dimer from (+)-catechin in organic solvent. These results suggest that this laccase has higher catalytic oxidation capacity of phenolic compounds in suitable organic media and favorite oligomers could be obtained.     

Catalysis by Laccase (From Coriolus uersicolor) in Microheterogeneous Media of the Water/Organic Solvent/Surfactant Type

Catalysis by laccase from Coriolus uersicolor solubilized in the ternary systems of surfactant/water/organic solvent type, namely, Aerosol OT/water/octane, Brij 56/water/cyclohexane and egg lecithin/water/octane + pentanol + methanol mixture, has been studied. The laccase activity is found to depend, in principle, not only on the water/surfactant molar ratio, but on the surfactant concentration (with its hydration degree being constant) as well. The following inferences should be emphasized. Firstly, in all the systems under study, the catalytic activity (k_cat) of laccase entrapped into surfactant reversed micelles increases more than 50 times (when the surfactant concentration is extrapolated to zero) compared with the k_cat value in aqueous solution. Secondly, the catalytic activity (k_cat) of laccase entrapped in hydrated Aerosol OT aggregates, having lamellar, reversed cylindrical (hexagonal) and reversed micellar structure, depends greatly on the aggregate type. In other words, the phase transitions, i.e. an alteration in the packing of hydrated Aerosol OT molecules, evokes a sharp reversible change in the enzymatic activity. Thirdly, in the same phase, the catalytic activity of the solubilized enzyme depends on the linear dimensions of water cavities inside the surfactant aggregates (i.e. on the water content in the system under study). All these effects, regulating enzymatic activity, are probably caused by an alteration of the conformational mobility of laccase molecules incorporated into the inner polar cavities inside the surfactant aggregates.     

Reverse micelle-based chemiluminescence system for quinine sulphate.

A new chemiluminescence (CL) method is proposed for the determination of quinine sulphate, which is based on the dichloromethane solvent extraction of the ion-pair complex of tetrachloroaurate (III) with quinine sulphate, and luminol CL detection in a reversed micellar medium formed by the cation surfactant cetyltrimethylammonium bromide in a dichloromethane-cyclohexane (3:7, v/v)-water (0.35 mol/L Na2CO3 buffer solution, pH 11.5). The ion-pair complex of tetrachloroaurate (III) with quinine sulphate produced an analytical CL signal when it entered the reversed micellar water pool. In optimum conditions, CL intensities are proportional to the concentrations of the studied drug over the range 0.015-10 microg/mL, with a detection limit of 1.5 ng/mL. The relative standard deviation (RSD) is 1.38% for 2.5 microg/mL quinine sulphate (n=11). The method has been applied to the determination of the studied drug in biological fluids, with satisfactory results.     

Purification and characterization of UDPG:ginsenoside Rd glucosyltransferase from suspended cells of Panax notoginseng

Heterogeneity of ginsenosides is an interesting and important issue because those structure-similar secondary metabolites have different or even totally opposite pharmacological activities. In this work, a new enzyme UDP-glucose:ginsenoside Rd glucosyltransferase (UGRdGT), which catalyzes the formation of ginsenoside Rb₁ from ginsenoside Rd [Biotechnol. Bioeng. 89: 444–52, 2005], was purified approximately 145-fold from suspended cells of Panax notoginseng with an overall yield of 0.2%. Purification to apparent homogeneity, as judged by SDS-PAGE, was successfully achieved by using sequential ammonium sulphate precipitation, anion-exchange chromatography and native PAGE. The enzyme had a molecular mass of 36 kDa, and its activity was optimal at pH 8.5 and 35 °C. The enzyme activity was enhanced by Mn²⁺, Ca²⁺ and Mg²⁺, but strongly inhibited by Zn²⁺, Hg²⁺, Co²⁺, Fe²⁺ and Cu²⁺. The apparent K_m value for UDP-glucose and ginsenoside Rd was 0.32 and 0.14 mM, respectively. The biotransformation yield from ginsenoside Rd to Rb₁ by UGRdGT in 50 mM Tris–HCl buffer at pH 8.5 and 35 °C was over 80%. This work provides a basis for further molecular study on the ginsenoside Rb₁ biosynthesis by P. notoginseng cells and it is also useful for potential application to in vitro biotransformation from ginsenoside Rd to Rb₁.     

Lipase kinetics in organic-water solvent with amphipathic substrate for chiral reaction

Lipase from Pseudomonas cepacia was used for asymmetric hydrolysis of the substrate (+/-)1-chloro-2-acetoxy-3-(1-naphthyloxy)-propane, which is a precursor for (S)-(-)-beta-blocker synthesis. Because this substrate is insoluble in water and partially soluble in hydrophobic solvents such as hexane and octane, a mixture of hydrophilic organic solvents and aqueous buffer was used to study the initial reaction rates. Because of the amphipathic nature of the substrate, it can remain in three different forms: (1) monomeric (solution); (2) micellar; and (3) emulsion, depending on the acetone and substrate concentrations in the medium. This behavior is presented in a phase diagram. The enzyme was found to be active with micelle as well as emulsion form of the substrate, whereas it showed negligible activity with the monomeric form. Michaelis-Menten constants were determined experimentally for the emulsion and micellar part of the substrate. The initial rate of hydrolysis (v(0)) goes through a maximum with respect to the acetone content of the mixture. It is due to the combined effect of various factors occurring simultaneously with the increase in acetone content in the solvent. These phenomena are discussed based on the interfacial activation of lipase, deactivation of the enzyme at very high acetone concentration, and increase in critical micelle concentration (CMC) and critical emulsion concentration (CEC) with the increase in acetone content in the solvent. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 399-407, 1997.     

Reversed micellar extraction of trypsin: Effect of solvent on the protein transfer and activity recovery

By using trypsin as the model protein and AOT as the model surfactant, the effect of a variety of solvents on protein transfer and activity recovery during the liquid-liquid reversed micellar extraction was investigated. It was found that several solvents, including isooctane, octane, heptane, and kerosene, had a similar effect on the recovery of trypsin activity after a full cycle of forward and backward extraction, and could all be used as the solvents for AOT-reversed micelles in trypsin extraction. Two other solvents (hexane and cyclohexane), however, were not so efficient. (c) 1995 John Wiley & Sons, Inc.     

Stability of glucose 6-phosphate dehydrogenase complexed with its substrate and/or cofactor in aqueous and micellar environment

Inactivation of glucose 6-phosphate dehydrogenase (G6PDH) complexed with its substrate, glucose 6-phosphate (GP), and/or cofactor, NADP+, has been studied within the range 20-40 degrees C in three media: (a) 0.04 M NaOH-glycine buffer (pH 9.1); (b) Aerosol OT (AOT) reversed micelles in octane; and (c) Triton X-100 micelles in octane supplemented with 10% hexanol. The enzyme inactivation was characterized quantitatively by first order rate constants, kin (s-1). In the case of G6PDH-NADP+ complexes, the values of kin were independent of the initial concentrations of G6PDH, either in aqueous medium or AOT micelles. The values of kin for the complex G6PDH-GP were inversely related to the initial concentration of the enzyme, in both aqueous and micellar media. When inactivation of both complexes were studied in AOT micelles, minimum values of kin corresponded to the degree of hydration W0 = 16.7; at W0 > 16.7 and W0 < 16.7, kin increased. Within the range 20-40 degrees C, the values of kin measured for both complexes in aqueous medium were significantly lower than those measured in AOT micelles. Temperature dependences of kin were characterized by inflections in Arrhenius plots, which corresponded, depending on the medium, to certain temperatures from 33.6 degrees C to 40 degrees C. In all media studied, NADP+ complexes of the enzyme exhibited higher stability than their GP counterparts. The parameters of G6PDH and G6PDH-NADP+ melting, measured by differential scanning microcalorimetry (maximum temperature and half-width of the transition, enthalpy of denaturation, and van't Hoff enthalpy), provided unequivocal evidence of the higher stability of the complex as compared to that of the enzyme. In addition, this approach demonstrated that G6PDH undergoes destabilization in AOT micelles.     

Induction of toluene oxidation activity in Pseudomonas mendocina KR1 and Pseudomonas sp. strain ENVPC5 by chlorinated solvents and alkanes.

Toluene oxidation activity in Pseudomonas mendocina KR1 and Pseudomonas sp. strain ENVPC5 was induced by trichloroethylene (TCE), and induction was followed by the degradation of TCE. Higher levels of toluene oxidation activity were achieved in the presence of a supplemental growth substrate such as glutamate, with levels of activity of up to 86% of that observed with toluene-induced cells. Activity in P. mendocina KR1 was also induced by cis-1,2-dichloroethylene, perchloroethylene, chloroethane, hexane, pentane, and octane, but not by trans-1,2-dichloroethylene. Toluene oxidation was not induced by TCE in Burkholderia (Pseudomonas) cepacia G4, P. putida F1, Pseudomonas sp. strain ENV110, or Pseudomonas sp. strain ENV113.