Small-angle X-ray scattering analysis of stearic acid modified lipase.

Stearic acid modified lipase (from Rhizopus japonicus) exhibited remarkable interesterification activity in n-hexane, but crude native lipase did not. The structure of the fatty acid modified lipase had not been analyzed until now. We analyzed the modified lipase by small-angle X-ray scattering (SAXS) measurements in order to clarify the structure. SAXS measurements showed that the modified lipase consisted of a lipid lamellar structure and implied that the lipase was incorporated into the lamellar structure of stearic acid. The long spacings in the lamellar structures of the modified lipase and stearic acid were measured.

Preparation and catalytic performance of surfactant-manganese peroxidase-Mn(II) ternary complex in organic media.

A novel preparation method for surfactant-MnP-Mn(II) ternary complex utilizing water-in-oil emulsions has been developed. The surfactant-MnP complex was spectroscopically characterized, strongly suggesting that the heme environment of the surfactant-MnP complex in benzene is identical to that of native MnP in the aqueous buffer. o-Phenylenediamine oxidation catalyzed by the surfactant-MnP-Mn(II) ternary complex was performed in benzene. The ternary complex efficiently catalyzed the oxidation, and the complex was catalytically stable. Kinetic experiments revealed that the reaction mechanism was as follows: MnP is oxidized by H(2)O(2) and the oxidized intermediate catalyzes the oxidation of Mn(II) to Mn(III) and the latter, after complexed with malonate, readily oxidizes o-PDA inside the complex. Thus, the organic substrate o-PDA, but not Mn(III), shuttled between the surfactant-MnP-Mn(II) ternary complex and organic solvent.

Surface imprinting polymers for the recognition of nucleotides.

A highly selective polymer has been prepared for the selective separation of nucleotides by the surface imprinting polymerization. A dialkyl quaternary ammonium chloride was effective as the functional molecule for recognizing the difference in the structure of nucleotides. Adsorptive behavior of the ionic species of the structural analogues, inosine-5'-monophosphoric acid (IMP) and guanosine-5'-monophosphoric acid (GMP), could be controlled by changing the pH condition. Surface imprinting polymers were prepared under different pH conditions; pH 9.0 and pH 8.5. The IMP-imprinted polymers exhibited higher template effect for IMP than for a structural analogue, GMP. A reference polymer prepared without the imprint molecule neither exhibit any selectivity to IMP nor to GMP. The adsorption behavior was quantitatively evaluated by the binding constants for the IMP-imprinted polymer. The imprinting polymer was found to recognize a small structural difference in nucleotides.

Catalytic activity of laccase hosted in reversed micelles.

Nanostructured reversed micelles induce a high laccase activity in organic solvents, because enzymes can maintain their highly dimensional structure in water pools of reversed micelles [RMs]. Laccase attracts considerable attention as a novel industrial enzyme due to its high capability to catalyze the oxidation of aromatic compounds. The catalytic activities of lyophilized laccase and laccase entrapped in RMs were compared using an oxidative reaction. Laccase hosted in an anionic RM effectively catalyzed the oxidative reaction in various organic solvents, while lyophilized laccase exhibited no such catalytic activity. To optimize the preparation and reaction conditions for laccase in RMs, we examined the effects of pH of water pools of RMs, the concentrations of both enzyme and surfactant for the preparation of RMs, the hydration ratio (Wo), and the reaction temperature on laccase catalytic activity in organic media. Laccase entrapped in RMs exhibited the highest catalytic activity in isooctane under the following conditions: bis-2-ethylhexyl sulfosuccinate sodium salt (AOT) of 100 mM, pH 6.0, Wo=40, and reaction temperature of 60 degrees C. Under the optimum conditions, environmental pollutants such as bisphenol A, 2,4-dichlorophenol and 2,4,6-trichlorophenol were effectively degraded in 3 h.

Important parameters affecting efficiency of protein refolding by reversed micelles.

Refolding of denatured RNase A as a model of inclusion bodies was performed by reversed micelles formulated with sodium di-2-ethylhexyl sulfosuccinate (AOT) in isooctane. In the novel refolding process, a solid-liquid extraction was utilized as an alternative to the ordinary protein extraction by reversed micelles based on a liquid-liquid extraction. First, the effects of operational parameters such as concentration of AOT, W(o) (= [H(2)O]/[AOT]), and pH were examined on the solubilization of solid denatured proteins into a reversed micellar solution. The solubilization was facilitated by a high AOT concentration, a high W(o) value, and a high pH in water pools. These conditions are favorable for the dispersion of the solid protein aggregates in an organic solvent. Second, the renaturation of the denatured RNase A solubilized into the reversed micellar solution was conducted by addition of glutathione as a redox reagent. A complete renaturation of RNase A was accomplished by adjusting the composition of the redox reagent even at a high protein concentration in which protein aggregation would usually occur in aqueous media. In addition, the renaturation rates were improved by optimizing water content (W(o)) and the pH of water pools in reversed micelles. Finally, the recovery of renatured RNase A from the reversed micellar solution was performed by adding a polar organic solvent such as acetone into the reversed micellar solution. This precipitation method was effective for recovering proteins from reversed micellar media without any significant reduction in enzymatic activity.

Formation of biocompatible reversed micellar systems using phospholipids.

Formation of reversed micellar systems using biocompatible components was revealed by a significant increase of water content in the organic phase. Soybean lecithin (SL), which is a mixture of different phospholipids, and phosphatidylcholine (PC) purified from soybean were used as the amphiphilic molecule. Fatty acid and fatty acid ethyl esters were used as the organic solvent. Reversed micelles were formed in the following combinations of (amphiphilic molecule)/(organic solvent): SL/ethyl caproate, SL/ethyl oleate, SL/ethyl linoleate, PC/ethyl caproate, and PC/oleic acid. Characterization of the micelles using small angle X-ray scattering analysis was presented. Reversed micelles formed in SL/ethyl caproate, SL/ethyl oleate, and PC/ethyl caproate systems were spherical. Their radius of gyration was about 40Å when the water concentration in the organic phase was maximal. Maximal water concentrations in SL/ethyl caproate and PC/ethyl caproate reversed micellar systems decreased with increasing salt concentration in the aqueous phase. Micelle sizes also decreased with increased salt concentration. The extraction of protein cytochrome c using the reversed micellar system was demonstrated. Application of these reversed micellar systems will expand to pharmaceutical and food industries.

Surfactant-lactoperoxidase complex catalytically active in organic media.

A surfactant-lactoperoxidase (LPO) complex catalytically active in organic solvents was developed by the emulsion coating method. The oxidation of 2,6-dimethoxyphenol (2,6-DMP) was conducted by the surfactant-LPO complex in organic media. The LPO complex efficiently catalyzed the oxidation of 2,6-DMP in various organic solvents, although lyophilized LPO did not display the catalytic activity at all. To optimize the preparation and reaction conditions for the surfactant-LPO complex, we examined the effects of pH value in the water pools of W/O emulsions, kinds of oxidants, and the nature of organic solvents on the oxidation reaction. Its optimum activity was obtained when the pH value of the aqueous enzyme solution was adjusted to ca. 8 at the preparation stage. The LPO complex exhibited the highest catalytic activity in chloroform when H(2)O(2) was employed as the oxidant. Furthermore, the storage stability of the surfactant-LPO complex was far better than that of the surfactant-horseradish peroxidase complex. This high storage stability of the LPO complex will be a benefit for industrial usage of peroxidases.

Characterization and catalytic property of surfactant-laccase complex in organic media.

The oxidation of o-phenylenediamine catalyzed in anhydrous organic solvents by surfactant-laccase complex was investigated. The complex was prepared by utilizing a novel preparation technique in water-in-oil (W/O) emulsions. The surfactant-laccase complex effectively catalyzed the oxidation reaction in various dry organic solvents, while laccase, lyophilized from an aqueous buffer solution in which its activity was optimized, exhibited no catalytic activity in nonaqueous media. To optimize the preparation and reaction conditions for the surfactant-enzyme complexes, we examined the effects of pH in the water pool of W/O emulsions, the concentration of enzyme and surfactant at the preparation stage, and the nature of organic solvents at the reaction stage on the laccase activity in organic media. Surfactant-laccase complex showed a strong pH-dependent catalytic activity in organic media. Its optimum activity was obtained when the complex was prepared at a pH of about 3. Interestingly, native laccase in an aqueous buffer solution exhibited an optimum activity at the same pH of 3. The optimum preparation conditions of surfactant-laccase complex were [laccase] = 0.8 mg/mL and [surfactant] = 10 mM, and the complex showed the highest catalytic activity in toluene among nine anhydrous organic solvents. The effect of a cosolubilized mediator (1-hydroxybenzotriazole (HBT)) on the reaction was also investigated. The addition of HBT at the preparation stage of the enzyme complex did not accelerate the catalytic reaction because HBT was converted to an inactive benzotriazole (BT) by laccase. However, the addition of HBT at the reaction stage enhanced the catalytic performance by a factor of five compared to that without HBT.

Quantitative determination of cultured strawberry-cell heterogeneity by image analysis: effects of medium modification on anthocyanin accumulation.

Cultured plant cells are often highly heterogeneous in terms of secondary metabolite production. We have developed a quantitative determination method that uses an image processing system to estimate such individual cell characteristics as content of the secondary metabolite, anthocyanin. In this study, strawberry cells producing anthocyanins were grown in modified Linsmaier-Skoog medium. Anthocyanin accumulation profiles of individual cells depended on medium compositions and were quantitatively determined using the new method. The modified medium supplemented with riboflavin and high sugar concentration showed a markedly higher anthocyanin accumulation profile and pigmented cell ratio than the other modified media. The maximum content was about 11mg (g-fresh cell weight)(-1), which was three times higher than that in the control medium. Moreover, the anthocyanin accumulation profiles in the individual cells cultured in all modified media could be approximated to the parts of the normal distribution curves with the constant variance.

Intermittent light irradiation with second- or hour-scale periods controls anthocyanin production by strawberry cells*

Anthocyanin production by strawberry cells depends not only on light intensity but also on the light/dark cycle operation with hour- or second-scale periods. These findings are useful for designing and operating photobioreactors for enhanced anthocyanin production. Intermittent illumination with a second-scale period produces the same amount of anthocyanin as continuous light, suggesting that the light intensity distribution within a photobioreactor does not cause suppressed production. In the hour-scale cycle, continuous light operation enhanced anthocyanin production more than the light/dark cycle process.

Surfactant-protease complex as a novel biocatalyst for peptide synthesis in hydrophilic organic solvents*

The peptide synthesis from N-acetyl-L-phenylalanine ethyl ester with alaninamide catalyzed by a surfactant-protease complex has been performed in anhydrous hydrophilic organic solvents. Proteases derived from various sources were converted to surfactant-coated complexes with a nonionic surfactant. The surfactant-subtilisin Carlsberg (STC) complex had a higher enzymatic activity than the other protease complexes and the initial reaction rate in tert-amyl alcohol was 26-fold that of STC lyophilized from an optimum aqueous buffer solution. Native STC hardly catalyzed the same reaction. The addition of water to the reaction medium activated the lyophilized STC, however, the reaction rate was much lower than that of the STC complex, and a hydrolysis reaction preferentially proceeded. The STC complex exhibited a high catalytic activity in hydrophilic organic solvents (e.g. tertiary alcohol). The addition of dimethylformamide as a cosolvent improved the solubility of amino acid amides and further activated the STC complex due to the water mimicking effect. When hydrophilic amino acid amides were employed as an acyl acceptor, the peptide formation proceeded efficiently compared to that using hydrophobic substrates. The surfactant-STC complex is a powerful biocatalyst for peptide synthesis because the STC complexes display a high catalytic activity in anhydrous hydrophilic organic solvents and did not require the excess amount of water. Thus the side (hydrolysis) reaction is effectively suppressed and the yield in the dipeptide formation is considerably high.

Analysis of pigment accumulation heterogeneity in plant cell population by image-processing system.

Plant-cultured cells are often highly heterogeneous in secondary metabolite productivity. The industrial application for large-scale metabolite production requires establishment of a stable high-producing cell line. In this study, image analysis of the individual cell is investigated as a method for evaluation of a heterogeneous cell population, and compared with the conventional method of estimation, which is based on average-cell productivity. Among strawberry cells producing anthocyanins, cells with a wide-range of pigment concentration were observed and maximum anthocyanin content was 10 times higher than the average value. In addition, a change of the frequency distribution was revealed in batch cultivation.

Refolding of cytochrome c using reversed micelles.

A novel protein refolding method using reversed micelles has been developed, which could replace the conventional dilution method using a buffer solution. The novel refolding method enables efficient refolding at a high protein concentration. In the present study, denatured bovine heart cytochrome c was directly solubilized in AOT reversed micelles using the solid-liquid extraction technique. Results reveal that addition of urea in small amounts facilitates solubilization of denatured protein into the reversed micellar phase. Reversed micelles containing a high concentration of denatured cytochrome c could be easily prepared by the novel solubilization method. The nanostructural environment formed by the surfactant molecules in organic media is considered to promote the renaturation of denatured proteins because the protein molecules are isolated from each other through the solubilization step. Although the recovery of entrapping proteins from the reversed micellar phase was known to be difficult in a conventional reversed micellar extraction operation, the addition of alcohol to the recovery phase improved the efficiency of back extraction. Therefore, we succeeded in recovering renatured cytochrome c from the reversed micelles. We demonstrated that the novel protein refolding method is very useful for the renaturation of denatured proteins.

Surfactant-histidine-heme ternary complex as a simple artificial heme enzyme in organic media

A surfactant-heme complex which shows peroxidase activity in organic media has been prepared by a method utilizing water-in-oil (W/O) emulsions. Both the aqueous phase pH and the type of surfactant appeared to have prominent effect on the catalytic activity of the heme complex in benzene. The catalytic efficiency of the heme complex was enhanced more than ten times by adding histidine to the aqueous phase of W/O emulsions in the preparation process. The enhancement of peroxidase activity was observed only in a nonaqueous medium due to the increase of the effective concentration of histidine as an activator. In the present study, we propose a simple preparation method for an artificial heme enzyme which works in nonaqueous media. Copyright 1999 John Wiley & Sons, Inc.

Protein separation using affinity-based reversed micelles

Reversed micellar two-phase extraction is a developing technique for protein separation. Introduction of an affinity ligand is considered to be an effective approach to increase the selectivity and capacity of reversed micelles. In this article, Cibacron Blue F3G-A (CB) as an affinity ligand was immobilized to reversed micelles composed of soybean lecithin by a two-phase reaction. The affinity partitioning of lysozyme and bovine serum albumin (BSA) to the CB-lecithin micelles was studied. Formation of mixed micelles by additionally introducing a nonionic surfactant, Tween 85, to the CB-lecithin micelles was effective to increase the solubilization of lysozyme due to the increase of W0 (water/surfactant molar ratio)/micellar size. The partitioning isotherms of lysozyme to the CB-lecithin micelles with and without Tween 85 were expressed by the Langmuir equation. The dissociation constants in the Langmuir equation decreased on addition of Tween 85, indicating the increase of the effectiveness of lysozyme binding to the immobilized CB. On addition of 20 g/L Tween 85 to 50 g/L lecithin/hexane micellar phase containing 0.1 mmol/L CB, the extraction capacity for lysozyme could be increased by 42%. Moreover, the CB-lecithin micelles with or without Tween 85 showed significant size exclusion for BSA due to its high molecular weight. Thus, lysozyme and BSA were separated from artificial solutions containing the two proteins. In addition, the affinity-based reversed micellar phase containing Tween 85 was recycled three times for lysozyme purification from crude egg-white solutions. Lysozyme purity increased by 16-18-fold, reaching 60-70% in the recycled use.

Intermittent light irradiation with a second-scale interval enhances caffeine production by coffea arabica cells

We developed novel equipment that intermittently illuminates Coffea arabica cell suspensions at a second-scale interval and investigated how intermittent irradiation enhances caffeine biosynthesis by C. arabica cells. The light/dark cycles consisting of 2 s of illumination and 18 s of darkness enhanced caffeine production, reaching the same level as for continuous light. The intermittent illumination increased the production efficiency regarding light consumption by a factor of 10. Caffeine production was determined by light intensity regardless of intermittent or continuous light irradiation. We propose a new concept for designing a photobioreactor that is applicable to secondary metabolite production by plant cell culture.

Affinity extraction of proteins with a reversed micellar system composed of Cibacron Blue-modified lecithin.

Crude soybean lecithin was used as a novel surfactant to form reversed micelles in n-hexane. Cibacron Blue F-3GA (CB) was directly immobilized to the reversed micelles by a two-phase reaction. The reversed micellar system without CB showed low solubilizing capacity for low molecular weight proteins, lysozyme, and cytochrome c due to the weak electrostatic interactions. The introduction of CB significantly increased the solubilization of lysozyme because of its affinity binding to CB but showed no effect on the solubilization of cytochrome c since it did not bind to CB. Although bovine serum albumin had an affinity for CB, it was not extracted to the reversed micelles containing CB because its high molecular weight resulted in a significant steric hindrance effect. Thus the reversed micellar system had a high selectivity resulting from both biospecific and steric hindrance effects. The extraction yield of lysozyme decreased significantly with increasing ionic strength. Therefore, the back extraction of lysozyme was carried out using a stripping solution with an ionic strength of 0.865 mol/L. The overall recovery yield of lysozyme after back extraction could be increased to 87% by stripping for 2 h. The recovered lysozyme exhibited an activity equivalent to native lysozyme, and its secondary structure was also unchanged.

Capture of microbial cells on brush-type polymeric materials bearing different functional groups.

A brush-type microbial-cell-capturing polymeric material was prepared by radiation-induced grafting of an epoxy-group-containing monomer, glycidyl-methacrylate (GMA), onto a polyethylene-based fiber. The epoxy ring (EO) of GMA was opened with different degrees of introduction of diethylamine (DEA). The residual epoxy group was hydrophilized by ethanolamine (EA). The prepared DEA membranes with coexisting EO or EA groups were tested for their ability to capture Staphylococcus aureus and Escherichia coli cells. The DEA membrane (2.7 mol/kg of product of DEA group density) with coexisting EO groups (DEA-EO membrane) exhibited good S. aureus-cell-capturing ability with a capturing rate constant of 1.82 x 10(-6) m/s, whereas the DEA membrane with coexisting EA groups (DEA-EA membrane) retarded capturing abilities for both S. aureus and E. coli cells. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 523-528, 1997.

Taxol (paclitaxel) production using free and immobilized cells of Taxus cuspidata.

The production characteristics for Taxol (paclitaxel) using free and immobilized cells of Taxus cuspidata were investigated in a perfusion culture bioreactor. Although the cell growth was inhibited by higher dilution rates, the specific production rate of Taxol was increased by perfusion compared with that using batch operation. Perfusion cultures using a nylon-mesh cell separator for free suspension cells showed similar production profiles to those obtained using immobilized cells. Continuous Taxol production was successfully obtained at an approximate specific production rate of 0.3 mg/g DCW (dry cell weight) per day for up to 40 days. (c) 1997 John Wiley & Sons, Inc.