Recent studies of the structures and physical properties of crystalline fatty acids and triacylglycerols are reviewed, in an attempt to construct a molecular-level understanding of crystal structures and mixing phase behavior of the materials examined. In particular, polymorphism and mixing behavior of various cis-and trans-monounsaturated fatty acids and triacylglycerols have been discussed. One may draw a conclusion that acyl-acyl interactions through the unsaturated and saturated fatty acid moieties are critically important in determining the phase behavior of the mixture systems of the triacylglycerols, forming molecular compounds or supramolecules in a special case.
One long recognized feature of lipases is the phenomenon termed “interfacial activation”, which refers to the fact that they exert their maximum activities when the substrate concentration exceeds its critical micelle concentration. Little was known about the molecular basis of the lipase reaction mechanism including the interfacial activation until 1990 when highresolution crystal structures of human pancreatic and Rhizomucor miehei lipases were reported. Both enzymes were shown to have triads of Ser, His, andAsp, reminiscent of the catalytic triads of chymotrypsin and subtilisin. These putative catalytic centers were not exposed to the solvent, but were buried under surface loops, suggesting significant conformational changes before the catalytic event. This review assesses current progress in crystallographic studies of lipases, which lead to understanding of the structure-function relationships of the enzymes.
The classical concept of the static lipid-protein architecture of the biological membrane has been completely remodeled by the new concept of the fluid mosaic model proposed by Singer and Nicolson (1972). This new theory has provided many useful clues toward better understanding of the membrane functions. Membrane lipids are now recognized as precursors of bioactive molecules which are generated upon cell stimulation by agonists and act as second messengers produced by various phospholipases. It is thus well accepted that membrane lipids play crucial roles in signal transduction.
Several hundreds of sphingoglycolipids in nature have been identified so far, and their chemical structures and metabolism have been well elucidated. Their biological relevance especially in vivo is an important issue to be addressed. A growing body of evidence suggests that sphingoglycolipids are involved in various biological processes such as cellular proliferation, differentiation, and recognition. They are also identified to be receptors of several viruses and bacteria or antigen epitopes in some of auto-immune diseases. Recent advance of this research field is reviewed.
Erythrocytes undergo oxidative damage including membrane lipid peroxidation and protein oxidation during the aging process. Naturally occurring IgG autoantibody to band 3 glycoprotein of the erythrocyte membrane (anti band 3) binds to the aged erythrocytes. The IgG binding is a critical event in the removal of aged erythrocytes from the circulation since IgG-bound cells are destined to be phagocytosed by the macrophages in spleens. The selective recognition of the aged cells by the antibody occurs by the following successive mechanisms : 1) lipid peroxidation and/or SH oxidation of proteins in the membrane, 2) clustering of band 3 glycoprotein on the membrane, 3) anti-band 3 binding to the sialyl poly-N-acetyllactosaminyl saccharide chains of the clustered band 3. These events indicate that lipid peroxidation and protein oxidation are closely related to the erythrocyte aging and the mechanism of the aged cell removal from the circulation.
Lipids forming lamella are located in the intercellular spaces between stratum corneum of the skin and serve as barrier and water reservior for protecting the skin from foreign hazards and water loss. The lipids consist mainly of ceramides including acylceramides with linoleic acid in the ester linkage. Ceramides are an essential component for supporting lipid lamella where non-freezible, unbound water is compartmentalized, offering water-holding properties to the stratum corneum. This function was confirmed by recovery experiments on lipid-depleted stratum corneum combined with differential scanning calorimetry analysis. On the other hand, the acylceramides with linoleic acid play an crucial role in the barrier function by stabilizing the lamella as rivet. This role was demonstrated by recovery experiments in which only the linoleic acid bearing acylceramides had a potential of repairing barrier disrupture as seen in essential fatty acid deficiency rat skin and UV irradiated skin. Skin disease which suffers from a marked deficiency in both barrier and water-holding function is atopic dermatitis. Consistently, there is a marked decrease in the level of ceramides including acylceramides in the stratum corneum of patients with atopic dermatitis which can also be improved by the application of the these ceramide derivatives.
Unsaturation of fatty acids of glycerolipids in biological membranes can be altered by changing the growth temperature of the organism. Such temperature-induced changes in unsaturation of fatty acids are explained in terms of the regulation of membrane fluidity that is necessary for the proper functioning of biological membranes. Desaturases are responsible for the regulation of levels of the fatty acid unsaturation by introducing double bonds (unsaturation bonds) into fatty acids. In this review, we describe recent research on acyl-lipid desaturases and their importance in the tolerance and acclimation to cold of cyanobacteria. We include a discussion of the basic characteristics of fatty acid desaturation, the characterization of acyl-lipid desaturases, the genetic manipulation of desaturases in relation to modification of cold tolerance, and the temperature-regulated expression of genes for desaturases.
Lipase gene from Pseudomonas species is followed with an accessory gene called lipase activator gene, which is necessary for the production and excretion of mature lipase. The biochemical function of the activator gene has remained undetermined. Our group has isolated the activator gene, lipB, with lipase gene, lipA, from Pseudomonas aeruginosa TE 3285. Although lipA was expressed in Escherichia coli and its signal peptide was correctly processed at the same position as in Pseudomonas, the mature lipase deposited as inactive, inclusion bodies in E. coli cells. The lipase activity of the inclusion bodies did not recover by solubilization with urea. To gain lipase activity, solubilization required the fusion protein of lipB protein with glutathione S-transferase. The mode of the reactivation is different from chaperones and rather similar to intramolecular chaperones.
A marine bacterium, judged as a new species close to Shewanella putrefaciens, was isolated from the intestinal contents of the Pacific mackerel. The isolated strain SCRC-2738 prodused EPA as a sole polyunsaturated fatty acid amounting to 2540 % of the total fatty acid in the cells. EPA existed as phospholipids in the cell and was found in the sn-2-position of phosphatidyl ethanolamine and phosphatidyl glycerol. The physiological activity of the EPA-phospholipids extracted from SCRC-2738 cells was examined following administration to stroke-prone spontaneously hypertensive rats. After four weeks of oral administration, serum lipids (choresterol, triacylglycerol and phospholipid) and the systolic blood pressure were reduced significantly, compared to the control rats fed soybean lecithin. Moreover, we observed a significant reduction of the weight of perirenal and paraepididymal adipose tissues (2638 %). The 38 kbp genome DNA fragment was cloned from SCRC-2738 and expressed in Escherichia coli, which resulted in the production of EPA. The nucleotide sequence of the 38 kbp DNA fragment was determined. The DNA fragment contains eight open reading frames, and three of them possess homology with enzymes involved in fatty acid synthesis.
All of the archaea (archaebacteria), the extreme thermoacidophiles, the extreme halophiles, and the methanogens, exist in the harsh environment of the earth. Their membrane lipids are mainly composed of the derivatives of archaeol (sn-2, 3-diphytanylglycerol diether) and its dimer, caldarchaeol (dibiphytanyldiglycerol tetraether) along with variations in the polar head groups. The unique properties of the archaeal lipids can be used to taxonomically distinguish the archaea from eubacteria and eukaryotes and also delineate the subgroups of the archaea. The structure and composition of the lipids derived from archaeol and caldarchaeol or their variants found in the three domains of archaea are described. The uniqueness of the archaeal lipids has been used to study the following areas : the phylogenical and evolutional aspects of the membranes, biotechnological applications such as liposomes with remarkable thermostability, and drug delivery system. Computer simulation studies of the three-dimensional structure of the archaeal lipid are also presented.
Mycelia of a fungus Mortierella alpina 1S-4 and the mutants derived from it are new and rich sources of C20 polyunsaturated fatty acids (PUFAs) of pharmacologically and dietary importance. M. alpina 1S-4 producs a unique triacylglycerol with high n-6 PUFA content. The mycelial content of arachidonic acid reaches 274 mg/g dry mycelia (4.3 g/L) on cultivation of the fungus in a medium containing glucose and yeast extract. The value accounts nearly 70 % of the total fatty acids in the extracted oil. Mutants defective in A 5 desaturase are potent produces of dihomo-γ-linolenic acid (4.1 g/L). A Al2 desaturase-defective-mutant produces an oil containing only n-9 PUFAs. The mycelial Mead acid content is 141 mg/g dry mycelia (1.9 g/L). An oil rich in n-3 PUFAs can be produced by the same mutant when grown in a medium containing linseed oil. The content of total n-3 PUFAs (i.e., α-linolenic acid+8, 11, 14, 17-cis-icosatetraenoic acid+5, 8, 11, 14, 17-cis-icosapentaenoic acid) is nearly 50 % in total mycelial fatty acid. The enzyme systems for the biosynthesis of PUFAs in the Mortierella fungi is also discussed.
Docosahexaenoic acid (DHA) and icosapentaenoic acid (EPA) are valuable nutrients because they have important roles as the constituents of structural lipids in the nervous tissues of human brain and retina. In addition, DHA, EPA and other omega-3 fatty acids are important factors in the prevention of several human diseases. Fishes have been the dietary source of these fatty acids and they originate from the marine microbes upon which the fishes feed. Therefore, these marine microbes cultured under control could be utilized as alternative source of the omega-3 fatty acids. Marine microalgae are rich in DHA and EPA. However, very few species of freshwater algae contain significant amounts of these compounds. The marine unicellular alga Isochrysis galbana is well known as a source of such fatty acids and this species has been used extensively as a food source for aquaculture. The advantage of the marine microalgae such as I. galbana over other microbes as sources of polyunsaturated fatty acid is that this alga may be grown photosynthetically using carbon dioxide as the sole carbon source and seawater as a source of minerals. In this paper, we focused on the production of marine microalgae as an alternative source of polyunsaturated fatty acid. We have screened a number of species of marine microalga specifically for their ability to produce DHA and EPA and have optimized the growth conditions for I. galbana. The utilization of microalgae as feed for cultivation of rotifers is also reported. Moreover, the applications of genetic engineering for EPA production are also demonstrated.
Docosahexaenoic acid (DHA) has recently attracted nutritional attentions through its specific function in brain/retina as well as beneficial effects of (n-3) polyunsaturated fatty acids. Firstly, the physiological activity of DHA is reviewed from recent publications, and compared to the activity of icosapentaenoic acid (IPA), with respect to its incorporation into plasma/tissues, its anticoagulant/anti-inflammatory effect, its effect on brain/retina, its anticancer effect, and adverse effects of excess intake, if any. Secondly, reports on microbial lipid (single cell oil, SCO) production including DHA are reviewed, which include Crythecodinium cohnii, Thraustochytrium aureum, Vibrio marinus etc. Two trials for DHA-SCO using C. cohnii has revealed high cell/DHA productivities in a semicontinuous culture (50 g/L of cell mass) and a continuous culture (120 mg/L·h of DHA). A new strain, SR 21, isolated from the coral sea water in Yap Islands and identified as Thraustochytrium sp., showed tolerance to high concentration of glucose and to mechanical mixing and resulted in the highest DHA productivity at 4000 mg/L in a fermenter culture for 7 day.
Lipid-biomodification ability was examined for yeasts isolated from soil using culture medium containing beef tallow (2 %). Some yeasts, e.g. Candida, Trichosporon and Rhodotorula species were able to grow on fats and oils. Fatty acid and triacylglycerol compositions were modified in lipids of some strains. Candida sp. MIS-1 and YM1-1 preferentially produced oleic acid. Candida sp. MIS-1 had high level of triacylglycerol with a melting point like olive oil. Fatty acid composition of lipids in Candida lipolytica IAM4948 and Rhodotorula sp. A03-5 was similar to that of cacao butter. Yeast oils obtained from C. lipolytica provided the melting characterization different from beef tallow.
Secretive production of lipids, which are useful source for engine-driving fuel, by microbial process using carbohydrate biomasses as substrate has been invesigated in our laboratory. This review consists of four parts concerning breedings and selection of lipid-secretive microorganisms : (1) breedings of long-chain fatty aicds-secretive strains from Candida lipolytica L-1 by a step-wise mutagenesis process, (2) selection of a triacylglycerol (TG) -secretive and accumulative yeast strain Trichosporon sp. SH45Y, (3) breedings of mutants, SH45Y-derivatieves, having potent ability to produce TG secretively from glucose, a typical biomass charbohydrate, and (4) selection of microorganisms which produce liquid-n-alkane in culture medium by utilizing long-chain fatty acids and TG ; a strain of Penicillium decumbens can produce liquid alkanes with a yield of approximately 60 % in weight from palm kernel oil, a commercial TG.
Recent advances in genetic engineering make it possible to design lipids produced by oleaginous plants and microorganisms. Mechanism for lipid accumulation and lipid body formation is one of the most important knowledge for the basis of the lipid designing technology. Recently extensive studies have been focused on the molecular structure of lipid bodies in plants, which reveals lipid body specific proteins termed oleosin. In microorganisms, however, much less is known about the molecular structure of lipid bodies. This papar reviews the chemical composition, enzyme activities and formation mechanism of lipid bodies in oleaginous plants and microorganisms. Recent works on diacylglycerolacyltransferase and lipid bodies in an oleaginous fungus, Mortierella ramanniana var. angulispora, are also described. Future works would be dedicated to answer questions about how proteins and lipids are assembled and sorted into lipid bodies.
Practical solvent-free bioreactor systems using immobilized lipases have been constructed to convert palm oil to high quality foodstuff oil without quality deterioration through hydrolysis of triglycerides in oil. To avoid hydrolysis, moisture level of substrate oil has to be maintained at less than the solubility level of water in oil, which we call ultra-micro aqueous level. On the otherhand, lipase is well known to manifest its activities mostly at the interface between oil and water phases. To make lipase manifest its activities at the ultra-micro aqueous oil phase, the novel bioreactor systems with the new immobilizing method of lipase together with activator onto hydrophylic carriers, and without a drying procedure have been developed. These biochemical accomplishments show high promises for efficient convertion of edible fats and oils to highly valuable foodstuff, which can not be attained by means of chemical or physical methods.
Newly developed drugs have asymmetric atoms in their chemical structure, and biological effects reside ordinarily in only one of its enantiomers. Another enantiomer has different biological effects or toxicity. Their action on beings is competitive and differs in adsorption, metabolism, degradation and excretion. Therefore, it is important to establish the optical resolution process in the production of pharmaceuticals. The authors have developed the optical resolution system for the production of diltiazem intermediate and industrialized the hollow fiber type bioreactor with liquid-liquid contact mode. Diltiazem is useful as a coronary vasodilator and has been widely used in more than 100 countries in the world for 20 years. An asymmetric hydrolytic en zyme for trans-3- (4-mehoxyphenyl) glycidic acid methyl ester [(±) -MPGM], a keyintermediate in the synthesis of diltiazem, was found in Serratia marcescens. This lipase was immobilized on a spongy layer of the shell side of hollow fiber ultrafiltration membrane by means of physical adsorption. Asymmetric hydrolysis was carried out semicontinuously in membrane reactor circulating both toluene solution containing (±) -MPGM in the shell loop and aqueous solutioninthe lumen loop. Since reaction and product separation were achieved simultaneously, crystalline (-) -MPGM with a high yield and optical purity of 100 % ee was obtained through 7 repeated runs by concentration of the toluene phase after reaction. Tanabe Seiyaku has industrialized this system with a series of 50 m2 membrane bioreactor. The number of total steps for the synthesis of diltiazem reduced to 5 from 9, and the cost of production reduced 2/3.
High FFA rice bran oil was continuously converted to an oil containing more than 74 % of triacylglycerol (TG) by immobilized lipase. The synthesis with evaporation by heating was not suitable because of the increasing peroxide value of the oil. The process can be industrialized if the enzyme cost is reduced less than 510 % the value added by the synthesis. More than 95 % of polyunsaturated fatty acid (PUFA) or ethyl ester of the fatty acid such as docosahexaenoic acid (DHA) was converted to TG. Continuous lipolysis system is constructed consisting of a loop connecting a fixed-bed reactor containing immobilized lipase from Pseudomonas fluorescens biotype I and an oil-water separator. The feasibility of PUFA production was discussed by using the lipolysis reactor.
Various natural phospholipids which are difficult to be obtained from natural sources can be synthesized from lecithin by phopholipase D-catalyzed transphosphatidylation. It is also able to synthesize novel unnatural phopholipids which contain biologically active compounds such as vitamins at the polar heads of the phospholipids. Initinally phospholipase D from cabbage leaves were mostly used, but recently bacterial enzymes having high transphosphatidylation activities have been mainly employed. Lipase-catalyzed ester exchange and phospholipase A2-catalyzed ester synthesis can be used to introduce arbitrary fatty acid to phospholipids under some mild reaction conditions. Howev er, the efficiency of these enzymatic acyl exchange reactions are still low, and further research will be needed.
The cholestrol esterase and lipase having the activity for, β-position of glyceride have synthetic activities of sterol-fatty acid esters and also have the ester interchange activities. These reaction equilibrium of ester synthesis or ester interchange incline toward synthesis. Therefore the reaction proceed toward the synthesis even if the reaction system have many water. Because of this character, we are able to have some systems for sterol-fatty acid ester synthesis, the aqueous medium reaction system, the water containing organic solvent system and the organic solvent-water two-phase system. Sterols having a hydroxyl group at the three position are available as alcoholic components. The fatty acid components to be used include saturated fatty acids, unsaturated fatty acids, branched fatty acids, α-hydroxy fatty acids and dicarboxyl fatty acis having carbon atoms from three to twenty four and these esters of lower alcohol or glycerine. Each outline of the reaction systems for industrial utilization (i.e. the continuous phase separation system with counter current liquid?liquid extractor, the continuous synthesis system by immobilized enzyme filled in a column and the air-lift reactor and the membrane reactor) are described. The Air-lift reactor gave the many amounts of sterol ester per enzyme (ester/enzyme=1200 by weight) and high synthesis ratio of 99 % (wt %) at high substrates concentration of 69 % by continuous elimination of generated water with blowing nitrogen gas into the reaction mixture. These reaction systems have developped and have reached a level to be able to use for industrial scale.
The supercritical fluids (SCF) have several excellent solvent properties such as low viscosity, high diffusivity and high dependence of solubility on pressure and temperature. The critical temperature of carbon dioxide is 304.1 K, low enough to treat thermally labile biomaterials without decomposition, and CO2 is non-toxic to human beings. Supercritical carbon dioxide (SCCO2) have been used in extraction of flavor substance from hop and other flavors and also in removal of undesirable substances such as caffeine in coffee beans and in removal of residual organic solvent in powdery medical raw materials. The interest in the applications of SCF is now enlarged from extraction to the other areas such as chemical and enzymatic reactions, material processings, formation of fine particles etc. Most of the enzymes are stable in anhydrous SCCO2, consequently the lipase reactions in SCF have been studied in the examples of modification of the physical property of triacylglycerols, resolution of optical isomers, syntheses of flavor esters etc. In this review the characteristics of SCF as solvent of enzymatic reactions are summarized with the main results so far obtained for the lipase reactions in SCF.
Triacylglycerols have specific properties which are useful for processing and/or modification of food systems according to their polymorphism, hydrophobic/hydrophilic balance, digestibility, and so on. These phenomena are mainly governed by the structure. Interfacial properties of 3-butyryl-1, 2-dipalmitoylglycerol at O/W emulsion, specific anti-bloom effect of 2-oleoy1-1, 3 dibehenylglycerol and restriction of absorption for 2-stearoyl-1, 3-dihexanoylglycerol are good examples. In the present study, we intend to summarize the relation between the structure and their specific properties.
Since the first reports on X-ray crystallographic determination of the 3 D structure of two lipases in 1990, 3 D structures of many other lipases and their complexes with inhibitors have been clarified to elucidate the lipase reaction mechanism. An approach through protein engineering has also been playing an important role in such studies. Recent development of lipase applications in industry have created demand for new lipases having superior and specific functions, leading to the improvement or alteration of lipase functions by a tool of protein engineering. In this article, recent progress in protein engineering on lipases is reviewed, focusing on four wellstudied families of lipase, i.e.. pancreatic lipase, Rhizomucor miehei lipase from filamentous fungi, Geotrichum candidum lipase and lipases from Pseudomonas sp. An understanding of the molecular basis for the broad substrate specificity of lipases as well as the reaction mechanisms may lead to the design and manufacture of protein-engineered lipases having improved properties for industrial use.
Characteristics and their uses of polymerizable phospholipids were described. Most phospholipids usually form vesicles whereas one possessing diacetylenic group forms tubule. Polymerization is performed to stabilize vesicles for prolonged or controlled release of ingredients. Among them hemoglobin encapsulated liposome were developed for artificial red blood cells using conjugated diene type phospholipids. Polymerization profile, physical stability and in vivo safety tests such as hematological or immunological study were described. In addition to performing good physical stability, polymerized liposomal hemoglobin (ARC) show good compatibility with blood. Although ARC affected on immunological system for several days after injec tion, the change was temporary. Methacrylic type phospholipids were also studied as biocompat ible materials. Surface coated with copolymer (BMA) show less precipitation of blood cells or proteins after contact with whole blood. It seemed that polymerized materials containing phos phoryl choline group were well tolerated in livings. Many kinds of these products are expected on the market in the near future.