Tateo Suzuki

Synthesis of sugar fatty acid esters by modified lipase.

A simple synthesis of sugar fatty acid esters was developed in a nonaqueous solution using lipase modified by synthetic detergent. Esterification of sugar was accelerated by continuous removal of water from the reaction mixture with a molecular sieve. When glucose and palmitic acid (1:1 by mole) were used as the starting substrates, more than 90% of glucose was converted to its ester in this system. The resultant product was 6-O-palmitoylglucose. Other mono- or disaccharides were also esterified by the modified lipase with high yield. It was shown that the modified lipase might act as a catalyst for the synthesis of sugar fatty acid esters.

Antioxidative components, xanthone derivatives, inSwertia japonica Makino

Substances with antioxidative properties were obtained from an ether extract ofSwertia japonica Makino. Six active components of the extract were isolated and identified as methylbellidifolin, methylswertianin, swertianin, bellidifolin, norswertianin and desmethylbellidifolin. These six xanthone derivatives were shown to possess different antioxidant activities by chemiluminescent assay. The antioxidative activities of bellidifolin, norswertianin and desmethylbellidifolin were higher than those of butylated hydroxytoluene (BHT) and α-tocopherol. On autoxidation of methyl linoleate, bellidifolin had activity similar to that of BHT. On the basis of the data present on antioxidative properties and data reported on the mutagenicities of the xanthones, both activities were shown to give a good correlation.

Preparation of organic-solvent-soluble enzyme (lipase B) and characterization by gel permeation chromatography

To analyse lipase-catalysed reactions in organic media, a product was developed to aid both solubilization of lipase B and the preservation of its activity in organic solvents using a synthetic detergent, didodecyl glucosylglutamate, according to Okahatas modification method. By this improved method, >50% of the lipase B could be converted into a solvent-soluble enzyme, which was called organic-solvent-soluble lipase. The organic-solvent-soluble lipase was successfully separated by gel permeation chromatography (GPC) from the excess of free detergent. The lipase activity was eluted at the fraction corresponding to a molecular weight of 130 kD. The composition of the purified solvent-soluble lipase was also investigated. It was estimated that 153 ± 25 molecules of the synthetic detergent were attached to one enzyme molecule, and the molecular weight of the complex was calculated to be 131 kD ± 16 kD based on the composition ratio. This value agreed with the molecular weight determined by GPC. These results confirmed that the organic-solvent-soluble lipase could be chromatographed by the GPC method and that its activity was preserved, suggesting that the GPC might be a very useful technique for the purification of organic-solvent-soluble enzymes.

Reactive properties of the organic solvent-soluble lipase

In a previous report, the organic solvent-soluble lipase was prepared using a synthetic detergent, didodecyl glucosyl glutamate, and it was estimated that 150 +/- 30 molecules of the detergent were attached to one lipase molecule based on gel permeation chromatography and chemical analysis. In this paper, the reactivity of the organic solvent-soluble lipase was compared with that of the native lipase to study the effect of the surrounding detergent on the thermostability and enzymatic reactivity. The activity of the organic solvent-soluble lipase was preserved in the organic solvents up to a temperature of 50 degrees C as in the case of the native lipase in buffer (pH 7.0). The influence of the chain length of fatty acids of the substrate triacylglycerols on the hydrolysis activities was studied. The organic solvent-soluble lipase hydrolyzed triacylglycerols with longer chains more rapidly than the native lipase. The presence of Ca2+ at 0.1 mM stimulated the activity of the native lipase, whereas Ca2+ at a high concentration inhibited it. On the other hand, even at a low concentration, Ca2+ inhibited the activity of the organic solvent-soluble lipase. These results suggest that the detergent attached to the lipase molecule affected the reactive properties.

Effect of detergent attached to enzyme molecules on the activity of organic-solvent-soluble lipases

In our previous work, the organic-solvent-soluble enzyme (lipase B) was prepared in high yield using a synthetic detergent. Using this method, lipases from various origins have been solubilized in organic solvents with several kinds of detergent and the influence of detergent attached to the enzyme molecule on the reactivity of the organic-solvent-soluble lipase was investigated. The activities of the organic-solvent-soluble lipases have been compared in a homogeneous solvent system, tetrahydrofuran containing 20% buffer. It is shown that the hydrolysis rate of the organic-solvent-soluble lipase is affected by the nature of the surrounding detergent and that the organic-solvent-soluble lipase prepared from a detergent with a large hydrophobic region hydrolyses hydrophobic substrates more rapidly than that prepared from a detergent with a small hydrophobic region.

CONFORMATION ANALYSIS OF GLYCERIDES BY NUCLEAR MAGNETIC RESONANCE

Abstract The conformation of the glycerol moiety of five glycerides, tri-, 1,3-di-, 1,2-di-, 1-mono-, 2-monopalmitin, was studied by using proton nuclear magnetic resonance ( 1 H-NMR) method. The spectra of 2-monopalmitin in chloroform- d 1 , and in tetrahydrofuran- d 8 solutions showed the existence of the conformer, in which the OC α  C β  C γ , OC α C β O, O C β  C γ O and C α C β C γ O angle have gauche configuration. The increase in the polarity of the solution decreased the population of this gauche conformer. The activation energy for the rotational barriers for the C α C β and C β C γ were large enough to separate the spectra of this conformer from other conformers by 1 H-NMR. This gauche conformation of 2-monopalmitin may possibly be stabilized by the intramolecular hydrogen bonds in low polarity solutions. In the case of the other glycerides, this conformer was not observed. The fractional populations of the conformers of each glyceride were also analyzed by comparing the coupling constants of the proton in the glycerol moiety in several solutions.

Kinetics of organic solvent-soluble and native lipase

Organic solvent-soluble lipase was prepared with a synthetic detergent. The solvent-soluble lipase was presumed to be a complex of the enzyme and the detergent. To investigate the effect of the detergent attachment to the enzyme on the reaction properties, the kinetics of the solvent-soluble lipase and of the native lipase were estimated by using glycerides in a homogeneous solution of buffer and tetrahydrofuran. Analysis of the direct interaction between the enzyme and the substrate could serve to characterize the steric structure around the active site of the lipase. The Km values also differentiated the solvent-soluble lipase from the native lipase. These findings show for the first time that the detergent surrounding the enzyme molecule may affect not only the solubility of the enzyme, but its kinetics as well.

Partition of protein (lipase) through insoluble complex with amphiphiles

An insoluble complex of soluble protein (lipase fraction) and commercially available amphiphile was prepared. The extraction of protein from complex revealed that the protein interacted with amphiphile was specific. The capacity of a protein to form the insoluble complex depended on its solubility and on amphiphile used in preparing solution. It suggested that the commercially available amphiphiles might be effective for partitioning soluble protein.