Wakako Tsuzuki

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.

Intestinal epithelial cells absorb γ-tocotrienol faster than α-tocopherol

To elucidate the transepithelial transport characteristics of lipophilic compounds, the cellular uptake of tocopherol and tocotrienol isomers were investigated in Caco2 cell monolayer models. These vitamin E isomers formed mixed micelles consisting of bile salts, lysophospholipids, free fatty acid, and 2-monoacylglycerols, then the micelles were supplied to Caco2 cells. The initial accumulation of tocotrienol isomers in Caco2 cells was larger than those of corresponding tocopherol isomers. There was little difference among the cellular accumulations of four tocopherol isomers. These findings suggested that the difference between the molecular structures of the C16 hydrocarbon chain tail in tocopherol and tocotrienol was strongly responsible for the rapid epithelial transport into the Caco2 cells membranes rather than the difference in the molecular structures of their chromanol head groups. Furthermore, the secretion of α-tocopherol and γ-tocotrienol from Caco2 cells was investigated using Caco2 cells plated on a transwell. The time courses of their secretions from Caco2 cells showed that the initial secretion rate of γ-tocotrienol was also larger than that of α-tocopherol. To investigate the intestinal uptake of α-tocopherol and γ-tocotrienol in vivo, the mice were fed single doses of α-tocopherol or γ-tocotrienol with triolein. The γ-tocotrienol responded faster in plasma than α-tocopherol, although the maximal level of γ-tocotrienol was lower than that of α-tocopherol. This suggested that the intestinal uptake properties of administered α-tocopherol and γ-tocotrienol would characterize their plasma level transitions in mice.

Acyl moieties modulate the effects of phospholipids on β-carotene uptake by Caco-2 cells

The intestinal absorption of carotenoids is though to be mediated by the carotenoid assembly in mixed micelles, followed by its transfer into the enterocytes and subsequent secretion to the lymph as chylomicron particles. In the present study we investigated the effects of phospholipids and lysophospholipids with diverse fatty acyl moieties on the uptake of β-carotene solubilized in mixed micelles by Caco-2 cells. Compared with phospholipid-free mixed micelles (NoPL), those containing long-chain PC inhibited β-carotene uptake (16∶0, 18∶1-PC≅16∶0, 18∶2-PC<14∶0, 14∶0-PC≅16∶0, 14∶0-PC <16∶0, 16∶0-PC<NoPL). However, mixed micelles containing medium-chain PC enhanced β-carotene uptake (NoPL<8∶0, 8∶0-PC<12∶0, 12∶0-PC<10∶0, 10∶0-PC), and short-chain PC did not affect the uptake. Among the lysophosphatidylcholine (LysoPC) class, a marked increase of β-carotene uptake by medium-to-long-chain LysoPC was observed (NoPL<12∶0-LysoPC<14∶0-LysoPC<18∶1-LysoPC<16∶0-LysoPC), although short-to-medium-chain LysoPC (6∶0-LysoPC to 10∶0-LysoPC) did not affect β-carotene uptake. The long-chain 16∶0,18∶1-PC increased the β-carotene efflux from cells and drastically changed the β-carotene UV-visible absorbance spectrum, compared with those of NoPL micelles. The acyl moieties of long-chain PC may interact with the carotenoid in the micelle interior, shifting the β-carotene partition toward the micellar phase. Medium-chain PC and long-chain LysoPC, which have nearly equivalent hydrophobicities, may enhance β-carotene uptake through their interaction with the cell membrane.

Polar organic solvent added to an aqueous solution changes hydrolytic property of lipase.

For developing further uses of lipase as a biocatalyst, its hydrolytic activity toward some esters was investigated in a miscible solution composed of a buffer and a polar organic solvent. Twenty percent dimethylformamide, 35% dimethylsulfoxide, 15% 1,4-dioxane, 15% dimethoxyethane, and 2% diethoxyethane promoted the hydrolysis by a lipase from Rhizomucor miehei toward some hydrophobic substrates, 4-methylumbelliferyl oleate, 4-methylumbelliferyl palmitate, and monoolein. While hydrolysis by this lipase toward the substrates with a relatively weak hydrophobicity (4-metylumbelliferyl heptanoate and 4-methylumbelliferyl nanoate) was suppressed by these solvents. A fluorometric analysis showed that the polar organic solvent in the buffer induced some conformational change around a tryptophan residue of R. miehei lipase. In addition to the influence of the miscible solvent on the solubility of the substrates, the conformational change of the protein induced by the miscible solvent would also affect the reactive properties of the lipase. Adding a polar organic solvent to an aqueous solution will be an efficient method for changing hydrolytic performance of lipases.

cis-trans isomerization of carbon double bonds in monounsaturated triacylglycerols via generation of free radicals.

We investigated the heat-induced cis/trans isomerization of double bonds in monounsaturated lipids. When triolein (9-cis, 18:1) was heated around 180 degrees C, small amounts of isomerization products were obtained depending on the heating period. The heat-induced isomerization of triolein was considerably suppressed by the addition of different antioxidants or under nitrogen stream, and these additives simultaneously inhibited the thermal oxidation of double bonds in triolein. Therefore, an intermediate of the thermal oxidation reaction might be responsible for the heat-induced isomerization of the double bonds in triolein. The thermodynamics of the heat-induced isomerization of triolein (9-cis, 18:1) and trielaidin (9-trans, 18:1) were investigated using Arrhenius plot. The Arrhenius activation energies of cis double bonds in triolein and trans double bonds in trielaidin were 106 kJ/mol and 137 kJ/mol, respectively. The calculated internal rotational barrier heights of these double bonds were similar to those of the double bond of 2-butene radical and significantly lower than those of non-radicalized double bonds in 2-butene. These results suggest that heat-induced cis/trans isomerization of triolein and trielaidin occurs mainly through the formation of radical species, which are the intermediates produced during thermal oxidation. The activation energy difference between the two forms suggests that trans trielaidin radicals are more stable than cis triolein radicals. The high thermodynamic stability of the trans double bonds in lipid radicals would influence the population of cis and trans isomers in edible oils and contribute to slight accumulation of trans-18:1 isomers during heating or industrial processing.

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.

Acidolysis between Triolein and Short-Chain Fatty Acid by Lipase in Organic Solvents

Ten kinds of lipases were examined as biocatalysts for the incorporation of short-chain fatty acids (acetic, propionic, and butyric acids) into triolein in order to produce one kind of reduced-calorie structured lipids. Trans-esterification (acidolysis) was successfully done in n-hexane by several microbial lipases. Among them, lipase from Aspergillus oryzae was used to investigate the effects of incubation time, substrate molar ratio, and water content on acidolysis. Finally, more than 80% of triolein was incorporated by butyric acid (molar ratio of triolein to butyric acid, 1:10) in the dried n-hexane at 52 °C for 72 h. More than 90% of the products was monosubstituent, which was esterified with this short chain fatty acid at the 1-position of the glycerol moiety of triolein. These results suggest that A. oryzae lipase would be a powerful biocatalyst for the synthesis of low caloric oil, such as triacylglycerol containing a mixture of long- and short-chain aliphatic acids.

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.

Fluorimetric analysis of lipase hydrolysis of intermediate- and long-chain glycerides

For the purpose of deducing the digestive behavior of dietary fat in the digestive organs, a fluorimetric method for the measurement of hydrolysis by porcine pancreatic lipase was performed using intermediate- and long- acyl chain glycerides as substrates. Insoluble glycerides constituted by C10-C16 acyl chains were mechanically dispersed in 100% buffer and hydrolyzed by porcine pancreatic lipase. After the reaction, fatty acid released by the enzyme was extracted and its carboxyl group was fluorescently labeled with 9-bromomethylacridine. The 9-acridinylmethyl derivative of the fatty acid was separated and determined by HPLC. The sensitivity of this method was about 1000 times higher than that of the titrimetric method. Only 0.5 ng of porcine pancreatic lipase was sufficient for one routine assay. This assay method was successfully applied to investigate the enzymatic properties of porcine pancreatic lipase with respect to dietary lipids. The effects of some physiological factors concerned with lipid digestion, such as bile salt and colipase, on the lipase hydrolysis were also examined. The method established in the present study could contribute to a highly sensitive assay of some hydrolases containing lipase with regard to insoluble substrates.

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.