Saburo Fukui

Application of immobilized lipase to regio-specific interesterification of triglyceride in organic solvent

SummaryLipase from Rhizopus delemar was immobilized by entrapment with photo-crosslinkable resin prepolymers or urethane prepolymers or by binding to various types of porous silica beads. The immobilized lipase preparations thus obtained were examined for their activity in converting olive oil to an interesterified fat (cacao butter-like fat), whose oleic acid moieties at 1- and 3-positions were replaced with stearic acid moieties, in the reaction solvent n-hexane. Although all of the immobilized preparations exhibited some activity, lipase adsorbed on Celite and then entrapped with a hydrophobic photo-crosslinkable resin prepolymer showed the highest activity, about 75% of that of lipase simply adsorbed onto Celite. Entrapment markedly enhanced the operational stability of lipase.

Application of immobilized lipase to hydrolysis of triacylglyceride

SummaryLipase from Candida cylindracea was immobilized by entrapment with photo-crosslinkable resin prepolymers or urethane prepolymers, and by covalent binding or by adsorption to different types of porous inorganic or organic supports. All of the immobilized lipase preparations thus obtained showed some activity for hydrolysis of olive oil. Lipase entrapped with a hydrophobic photo-cross-linkable resin prepolymer exhibited the highest activity, which was about 30% of that of the free counterpart. Entrapment method enabled lipase to gain operational stability. Semicontinuous hydrolysis of olive oil using immobilized lipase was also accomplished in a packed-bed reactor with a recycling system. In this reactor, immobilized lipase was observed to have the sufficient activity and stability.

Substrate specificity of coenzyme B12-dependent diol dehydrase: Glycerol as both a good substrate and a potent inactivator☆

Abstract The substrate specificity of adenosylcobalamin-dependent diol dehydrase was further studied in detail using an enzyme preparation that appears homogeneous by ultracentrifugal and gel electrophoretical criteria. Besides 1,2-propanediol and 1,2-ethanediol, glycerol, 1,2- and 2,3-butanediol were found to serve as substrate for the enzyme, whereas 1,3-propanediol was not. Of the substrate analogs tested, glycerol displayed some striking features: it was dehydrated to β-hydroxypropionaldehyde with concomitant inactivation of the enzyme. Although the initial velocity with glycerol was comparable to that with 1,2-propanediol, the dehydration reaction ceased almost completely within 3 min accompanying rapid, irreversible inactivation of the holoenzyme. 1,2- and 2,3-Butanediol were converted to butyraldehyde and methyl ethyl ketone, respectively, at a rate much lower than that with 1,2-propanediol. 2,3-Butanediol is the only compound, other than 1,2-diols, known at present to show a considerable substrate activity.

Stereoselective esterification of dl-menthol by polyurethane-entrapped lipase in organic solvent

Abstract Stereospecific esterification of dl -menthol was studied by the use of immobilized lipase in an adequate water-saturated organic solvent system. Lipase from Candida cylindracea immobilized by entrapment with urethane prepolymers and 5-phenylvaleric acid as the acyl donor were chosen based on the stereoselectivity and the yield of l -menthyl ester. Water-saturated cyclohexane or isooctane was found to be the most suitable solvent system. Entrapment significantly enhanced the operational stability of lipase.

Development of microbodies in Candida tropicalis during incubation in a n-alkane medium

Development of microbodies in Candida tropicalis pK 233 was studied mainly by electron microscopical observation. The yeast cells, precultured on malt extract, scarcely contained microbodies and showed very low catalase activity. When the precultured cells were transferred to a n-alkane medium and incubated with shaking, the number of microbodies increased and concomitantly the activity of catalase was enhanced. That is, both the area ratio of microbodies in the cell and the ratio of microbodies to cytoplasm in area increased significantly during the utilization of n-alkanes for 8 hrs. Localization of catalase in the microbodies was demonstrated cytochemically by use of 3,3′-diaminobenzidine, but other organella in the cell, except for vacuoles appearing in the early growth phase and mitochondria, were not stained with this reagent. Microbodies seemed to grow by division. Biogenesis of microbodies in the yeast cells is also discussed.

Application of photo-crosslinkable resin to immobilization of an enzyme

The aim of this communication is to report a novel convenient method for entrapping enzymes or microbial cells using photo-crosslinkable resins. The technique consists of mixing a liquid photo-crosslinkable resin containing photo-sensitive functional groups located at a desired distance, an appropriate initiator and an enzyme solution or microbial cell suspension, followed by illumination with near-ultraviolet light for only a few minutes. This simple procedure produced tailor-made matrices in which enzyme molecules or microbial cells were successfully entrapped. Typical experiments performed with a photo-sensitive resin, polyethyleneglycol dimethacrylate of different chain. lengths and yeast invertase are described in this paper. Various criteria indicate that this new method is very useful for immobilization of enzymes and other biologically active macromolecules.

Transformation of steroids by gel-entrapped Nocardia rhodocrous cells in organic solvent

SummaryWhole cells of Nocardia rhodocrous were immobilized by entrapment with photo-crosslinkable resin prepolymers or urethane prepolymers of either hydrophilic or hydrophobic character, and their activity in converting 3β- hydroxy-Δ5 to corresponding 3-keto-Δ4 in an appropriate organic solvent system (e.g., a water-saturated mixture of benzene and n-heptane (1∶1 by volume)) was studied. Although both hydrophilic gel-entrapped cells and hydrophobic gel-entrapped cells could transform dehydroepiandrosterone to 4-androstene-3, 17-dione, the latter showed a significantly higher activity than the former. In the cases of the oxidation of cholesterol, β-sitosterol and stigmasterol to the corresponding 3-keto-Δ4-steroids, only hydrophobic gel-entrapped cells exhibited the catalytic activity.The cells entrapped in adequate hydrophobic gels had steroid transforming activity comparable to that of the free counterparts. The operational stability was improved by the entrapment, especially in the case of the transformation of dehydroepiandrosterone. The activity of the gel-entrapped cells was found to correspond closely to the partition coefficients of substrates between the gels and the external solvent.

Application of photo-crosslinkable resin prepolymers to entrap microbial cells. Effects of increased cell-entrapping gel hydrophobicity on the hydrocortisone Δ1-Dehydrogenation

SummaryAcetone-dried cells of Arthrobacter simplex, whose steroid Δ1 activity had been previously induced, were entrapped by the use of photo-crosslinkable resin prepolymers. When the hydrophobicity of the cell-entrapping gel was increased by mixing a hydrophobic prepolymer (main chain component; polypropyleneglycol) with a hydrophilic prepolymer (main chain component; polypropyleneglycol) with a hydrophilic prepolymer (main chain component; polyethyleneglycol) (up to 30%), the hydrocortisone to prednisolone conversion rate of the immobilized cells increased significantly, attaining approximately 20% of that of the free cells. A 10% addition of organic solvents, such as methanol, to the aqueous reaction mixture enhanced the solubility of the substrate greatly and to a lesser degree the reaction rate of the immobilized cells. The presence of an electron acceptor, phenazine methosulfate or 2,6-dichlorophenolindophenol, stimulated the steroid conversion of the entrapped as well as the free cells. The stability of the entrapped cells over repeated reactions was improved by immobilization.

Ultrastructure ofCandida yeasts grown onn-alkanes

Catalase activities of the cells growing onn-alkanes of various strains ofCandida yeasts wer markedly higher than those of the cells growing on glucose, ethanol or acetate. In connection with this, electron-microscopical studies revealed abundant appearance of specific microbodies having homogeneous matrix surrounded by single unit membrane in the hydrocarbon-growing cells. Localization of catalase activity in the microbodies, in addition to the mitochondria, was confirmed by cytochemical treatment of the cells with 3,3′-diaminobenzidine reagent.

Stereoselective hydrolysis of dl-menthyl succinate by gel-entrapped Rhodotorula minuta var. texensis cells in organic solvent

Summarydl-Menthyl succinate was successfully hydrolyzed stereoselectively by Rhodotorula minuta var. texensis cells entrapped within photo-crosslinked or polyurethane resin gels in water-saturated n-heptane. The hydrolyzed product was found to be pure l-menthol. The catalytic activity of the immobilized cells, especially those entrapped in urethane polymers, was far more stable than that of the free cells. The half-life of the polyurethaneentrapped cells was estimated to be 55–63 days in the organic solvent.