Mikio Kawagoe

Application of bubble column fermentors to submerged culture of Schizophyllum commune for production of l-malic acid

Abstract To investigate the applicability of bubble column fermentors to the submerged culture of mushroom mycelia, cultivation of Schizophyllum commune IFO-4928 for production of l -malic acid was carried out using a standard bubble column and an external-loop airlift column. Maximum yields of l -malic acid were attained at a superficial air velocity of 0.7 cm/s for the standard bubble column and 1.7 cm/s for the external-loop airlift column. The optimum temperature was 27°C for both types of column, and the optimum concentrations of both calcium carbonate and glucose were 50 g/l. The yield of l -malic acid under opimum conditions for the airlift column was larger than for the standard bubble column. From the comparison of the production rates of l -malic acid in flask culture, a jar fermentor, an airlift column, and a standard bubble column, the airlift bubble column was found to be most suitable for the culture of S. commune for l -malic acid production.

Protein extraction using sugar ester reverse micelles

In protein extraction, using the nonionic surfactant sugar ester DK-F-110, the critical micelle concentration (CMC) of a DK-F-110/isopropyl alcohol(IPA)/hexane system was found to be at a DK-F-110 concentration of 0.5 g dm -3 , indicating that the sugar ester reverse micelles could be formed in hexane. At concentrations higher than the CMC, cytochrome c was extracted into the DK-F-110 reverse micelles as judged from UV spectra of the DK-F-110/IPA/hexane solution after it was contacted with the aqueous protein solution. In extraction of cytochrome c using this system, forward extraction was found to be pH-dependent, with high extraction percentage being obtained at pH 8. The forward extraction percentage was reduced by an increase in the buffer concentration, and at a buffer concentration of 0.5 mol dm -3 was ca 25% as high as that of sodium bis(2-ethylhexyl) sulfosuccinate(AOT) systems. An optimal DK-F-110 concentration was found to give the maximum forward extraction percentage. Addition of alcohol, especially IPA, to the micellar organic phase enabled the highly backward extraction of cytochrome c to be achieved without the formation of insoluble aggregates. The esterification reaction rate by Rhizopus delemar lipase in the DK-F-110 reverse micellar system had a higher maximum value than that of AOT and lecithin systems.

Efficacy of guanidium salts in protein recovery from reverse micellar organic media

The efficacy of guanidium salts in the recovery of extracted lysozyme from aerosol-OT (AOT) reverse micellar organic phase was investigated. Adding guanidium salt at a low concentration as pretreatment reagent in the feed solution led to successful protein recovery, and the enzymatic activity of the recovered lysozyme was well maintained. Among the electrolytes tested, caotropic guanidine thiocyanate (GuHSCN) was the most effective in recovering lysozyme as well as in preserving its activity. The presence of guanidium salt in the micellar organic phase markedly lowered the water content, apparently by reducing or eliminating accompanying water arising from lysozyme solubilization. CD data showed that the α-helix content of the lysozyme in the micellar phase in the presence of dilute guanidium salt was smaller than that in a guanidium-free micellar phase. These results indicated that the guanidium salt expelled lysozyme molecules from the micro-interface of the reverse micelles into the hydrophilic micro-water pool.

Submerged culture of Tricholoma matsutake mycelium in bubble column fermentors

Submerged culture of Tricholoma matsutake mycelium was carried out using two bubble column fermentors, a standard bubble column and an external-loop airlift column. The effects of the aeration rate and column type on culture performance in terms of the mycelia morphology, glucose consumption, cell yield, and growth rate were investigated. Morphologically, three types of pellets-large spherical, small spherical and filamentous-were observed depending on the aeration rate. On the whole, the standard bubble column gave a higher cell yield and a better growth rate than the airlift type. The maximum cell yield and growth rate attained at a superficial air velocity of 0.38 cm/s were superior to those obtained in a flask culture, suggesting that the bubble column fermentor has the potential to be used for submerged culture of T. matsutake.

Purification and properties of malic enzyme from Pseudomonas diminuta IFO-13182

Abstract A malic enzyme from a cell-free extract of Pseudomonas diminuta IFO-13182 was purified to electrophoretic homogeneity by DEAE-Sepharose, Sephacryl, and Blue-Sepharose chromatographies. The purified enzyme required either NAD + or NADP + as a coenzyme. From the results of coenzyme specificity, the enzyme should be classified as l -malate: NAD + oxidoreductase (decarboxylating) [EC 1.1.1.39]. The purified enzyme was most active at pH 7.5 and 50°C and was stable in the pH range from 7.0 to 9.0. The isoelectric point was pH 4.3. Its molecular weight was 680,000 by COSMOSIL 5-Diol high performance liquid gel filtration on chromatography and 65,000 by SDS polyacrylamide gel electrophoresis. This indicates that the enzyme consisted of 10 subunits. The malic enzyme activity with NADP + was about twice that measured with NAD + .

Extraction of flexibly structured protein in AOT reverse micelles: the flexible structure of protein is the dominant factor for its incorporation into reverse micelles

Abstract The extraction of flexibly-structured protein in Aerosol-OT (AOT)/isooctane reverse micelles was investigated. A flexibly-structured lysozyme was prepared by reduction and carboxymethylation of the disulfide bonds in the lysozyme molecule. For a comparison, lysozymes whose surface hydrophobicity was modified by monoacylation of the amino groups were also used. The extraction rate of the flexibly-structured lysozyme into the micellar phase was greater than that of the native and monoacylated lysozymes, although the free energy change of the lysozyme prepared by breaking the disulfide bonds was smaller than that of the lysozymes whose surfaces were monoacylated. Viscosity measurement of the micellar organic phase containing the modified lysozymes indicated that extraction of the flexibly-structured lysozyme changed the micelle–micelle interaction, while measurement of the interfacial tension between the AOT/isooctane and protein aqueous systems showed the flexibly-structured lysozyme to be the most amphiphilic in character. These results indicated that the flexible structure of a protein was more dominant than its surface hydrophobicity for its incorporation into reverse micelles, and that it leads to greater micelle–micelle interaction.

Minimal AOT Concentration of Binary Protein Component in Reverse Micellar Extraction

The minimal AOT concentration of a binary proteinmixture containing cytochrome c and lysozyme was investigated, and was found to be smaller than the sum of the minimal AOT concentration of each protein obtained in single-protein systems, whatever the lysozyme molar fraction in the mixture. The water content,W0(=the water/amphiphile molarratio), of the reverse micellar organic phase under the binary minimal AOT concentration became maximum at a lysozyme molar fraction of 0.5. The apparent interfacial area per single head group of AOT in the binary protein system, estimated from viscosity data, was greater than that of a protein-free system, and became maximum at a lysozyme molar fraction of 0.5. The interfacial tension between the protein mixture aqueous solution and AOT/isooctane solution was lower than that for each single-protein aqueous solution. The binary protein mixture containing cytochrome c and lysozyme thus exhibited higher amphiphilic characteristics. These results indicated that the proteins behaved like amphiphilic molecules, especially when both of the proteins coexisted in the reverse micellar phase.

Hydrolysis Behavior of Triglycerides by Rhizopus delemar Lipase in Reverse Micellar Organic Solvents

The structure of a reverse micelle consists of an aqueous micro-domain facing the polar heads of the amphiphilic molecule that surrounds this core interacting with the bulk organic solvent, through the hydrophobic chains. The polar cores of the micelles have the ability to solubilize a significant amount of water. When the enzymes are microencapsulated, they are located in the interior aqueous phase of reverse micelles, and interacting with the micellar interface depending on the enzyme species. Enzymes have been traditionally used in aqueous media, but reverse micelles became attractive, especially, when substrates and/or products are hydrophobic and a low water content is desired. This applies specially to lipase reactions since the solubility of triglycerides is largely improved in organic solvents (Han and Rhee, 1986; Nagayama et al., 1996). Furthermore, since the minute size of water pools in reverse micelles on a nanometer scale, reverse micellar organic solvent offers enormous W/O interface area in an organic solvent. Owing to these features, the reverse micellar organic solvent is anticipated as reaction medium for industrial processes of lipase-catalyzed reaction using a hydrophobic substrate (Lee et al., 1998; Naoe et al., 2001).

Dynamico of a Mukiplate Gas-liquid Reactor

多段気液反応装置の動特性を検討するため, 総括反応速度および装置流動特性のプロセス変量依存性を同時に考慮して転達関数を求めた。実験的に求めた反応装置動特性と, 実験に伴う非線型性の効果を考慮して, この伝達関数から模擬した装置動特性とは, ほぼ一致する。また先に報告したこの種の装量に対する効果的な制御系の設計方法は, 本報告で求めた伝達関数により, その動特性を表わせる反応装置に対しても適用できる。