Lipase catalysed transesterification of castor oil

Summary Lipase catalysed transesterification of castor oil with n-butanol gives mono and diglycerides, and butyl esters. n-Butanol is found to inhibit this reaction. Its amount influences the reaction rate and product distribution. Increasing the enzyme quantity increases the reaction rate and alters the product distribution.     

Monogalactosyl and digalactosyl diglycerides from heterotrophic, hetero-autotrophic, and photobiotic Euglena gracilis

The lipid of Euglena gracilis, dark-grown in a complete medium, contained 2% galactose. The lipid of Euglena gracilis, light-grown in either a complete or an inorganic medium, contained 13-14% galactose. Pure monogalactosyl and digalactosyl diglyceride fractions, isolated by column plus thin-layer chromatography, contained 50% of the lipid-bound galactose of dark-grown cells, and 80% of that of light-grown cells. Molar ratios of monogalactosyl to digalactosyl compounds ranged from 2 to 3. The results show that galactosyl diglycerides, stored in large amount in light-grown cells, persist in small amount in the dark-grown cells. Fatty acids in both the monogalactosyl and the digalactosyl diglycerides were mainly of the 16- and 18-carbon varieties, with high proportions of trienes. The monogalactosyl diglycerides were rich in hexadecatetraenoic acid. Strictly photobiotic cells had twice as much hexadecadienoic and hexadecatetraenoic acids in their monogalactosyl diglycerides, and three times as much hexadecadienoic and octadecadienoic acids in their digalactosyl diglycerides as did illuminated cells grown in a complete medium. Dark-grown (obligate) heterotrophs contained galactosyl diglycerides with high percentages of monoenes. Great compositional variations in the galactosyl diglycerides are thus induced by light and also by nonlipid exogenous metabolites.     

Biosynthesis of monogalactosyl diglyceride by chloroplasts from spinacia oleracea and from some gramineae

Synthetic diglycerides which differed in unsaturation of fatty acids gave the same incorporation of [¹⁴C]galactose from UDP-[¹⁴C]galactose when added to acetone powders of spinach chloroplasts up to about 0·6 mg diglyceride/20 mg acetone powder. Diolein and the endogenous diglyceride isolated from the acetone extract of chloroplasts stimulated galactolipid biosynthesis to a similar extent. With all diglycerides used, monogalactosyl diglyceride was the main product with little accompanying synthesis of digalactosyl diglyceride. The radioactivity in the monogalactosyl diglyceride synthesized from UDP-[¹⁴C]galactose by whole chloroplasts was distributed widely among the monogalactosyl diglycerides with different fatty acid composition. It is concluded that the enzyme which catalyses the transfer of galactose from UDP-galactose to diglyceride is not specific for polyunsaturated diglycerides and that the polyunsaturated monogalactosyl diglycerides arise either by desaturation of the fatty acyl residues after monogalactolipid synthesis or by transacylation. Acetone powders of chloroplasts prepared from several Gramineae did not exhibit transferase activity although whole chloroplasts were active.     

Biosynthesis of mitochondrial phospholipids using endogenously generated diglycerides.

When isolated mitochondria or microsomes from rat liver were treated with phospholipase C, the incorporation of radioactive phospholipid precursors was markedly enhanced, presumably as a result of production of diglycerides by hydrolysis of endogenous phospholipids. Incorporation of CDP[14C]choline into lecithin in rat liver or BHK-21 mitochondria could be attributed to residual contamination from elements of the endoplasmic reticulum, with added diglycerides or with endogenous diglycerides produced by the phospholipase C treatment. A similar stimulation of [gamma32P]ATP incorporation into phospholipids was observed with exogenous or endogenous diglycerides, but the mitochondrial diglyceride kinase in either case was also related to the degree of microsomal contaminants. It was concluded that previous studies showing negligible capacity of mitochondria for lecithin biosynthesis de novo were not explainable on the basis of limited accessibility of added diglycerides, and that formation of phosphatidic acid by diglyceride kinase was not of significance in rat liver mitochondria.     

Molecular species analysis of mitogen-stimulated 1,2-diglycerides in fibroblasts. Comparison of alpha-thrombin, epidermal growth factor, and platelet-derived growth factor

Recent studies have implicated the hydrolysis of phosphoinositides and phosphatidylcholine in agonist-stimulated events. The potent mitogen, alpha-thrombin, stimulates the generation of diglycerides in a biphasic and sustained manner in IIC9 fibroblasts (Wright, T. M., Rangan, L. A., Shin, H. S., and Raben, D. M. (1988) J. Biol. Chem. 263, 9374-9380). Using measurements of radiolabeled headgroup release and molecular species analysis, we previously determined that alpha-thrombin generates diglycerides through the hydrolysis of both the phosphoinositides and phosphatidylcholine at early times (15 s), and at later times (greater than or equal to 5 min) through the hydrolysis of primarily, if not exclusively, phosphatidylcholine (Pessin, M. S., and Raben, D. M. (1989) J. Biol. Chem. 264, 8729-8738). In contrast, IIC9 fibroblasts respond to the mitogenic treatments of (a) alpha-thrombin following chymotrypsin pretreatment or (b) epidermal growth factor by increasing their levels of diglycerides in a monophasic and sustained manner (Wright, T. M., Rangan, L. A., Shin, H. S., and Raben, D. M. (1988) J. Biol. Chem. 263, 9374-9380). In this report, we have analyzed the molecular species of the diglycerides generated by these two different treatments and have also examined the lipid response of IIC9 fibroblasts to platelet-derived growth factor. Based on both the molecular species analyses and the release of radiolabeled head-groups, all three of these different mitogenic treatments generate diglycerides primarily through the stimulation of phosphatidylcholine hydrolysis. However, while similar, the molecular species profiles of the diglycerides generated by these three treatments are not identical to the molecular species profile of total cellular phosphatidylcholine. In addition, the molecular species profiles of the diglycerides generated by these three mitogenic treatments greatly resemble each other, with significant differences between any two profiles occurring in at most one molecular species. This finding differs from that seen with alpha-thrombin stimulation alone, where the molecular species profile of the diglycerides generated following 5 min of alpha-thrombin stimulation is nearly identical to the molecular species profile of total cellular phosphatidylcholine. These data support the possibility of hormone-sensitive phosphatidylcholine pools or selective diglyceride metabolism.     

Biosynthesis of galactolipids by enzyme preparations from spinach leaves

The pH optimum for galactolipid synthesis from UDP-galactose by spinach chloroplasts is 7.2 in Tris-HCl or phosphate buffer. The products include sterol glycosides, trigalactosyl diglyceride (tentatively identified), digalactosyl diglyceride, and monogalactosyl diglyceride in increasing order of quantity. The proportion of monogalactosyl diglyceride decreases and that of digalactosyl diglyceride increases as the pH is lowered. The galactolipid synthesis is quite resistant to elevated temperature; maximal incorporation of galactose from UDP-galactose was observed at 45 degrees C. The proportion of monogalactosyl diglyceride was greater at the higher temperatures. As much as 40% of the galactolipid-synthesizing capability of a spinach leaf homogenate is not sedimented by centrifugation for 60 min at 100,000 g. An acetone powder of spinach chloroplasts contains enzymes which catalyze galactolipid synthesis. This preparation is dependent on added diglycerides in order to make galactolipid, whereas the chloroplast preparation is not dependent on added diglycerides. Molecular species of diglycerides were compared as requirements for galactolipid synthesis. The requirement was satisfied best by the diglycerides of highest unsaturation. Methylation of the free hydroxyl of the diglyceride eliminated the effectiveness.     

Triglyceride Interesterification by Lipases: 2. Reaction parameters for the reduction of trisaturated impurities and diglycerides in batch reactions

A model system consisting of pure triolein and palmitic acid and LipozymeTM, an immobilized lipase (E.C. 3.1.1.3.). has been used to determine the effects of various reaction parameters on the reaction rate and the formation of by-products in the interesterification reaction. The goal was to minimize the level of diglycerides and eliminate trisaturated triglycerides at an endpoint chosen so that the results could be applied to the production of cocoa butter substitutes. The levels of diglycerides, which are essential reaction intermediates, and trisaturated glycerides, which are believed to be formed as a result of spontaneous acyl migration of mono- and diglyceride intermediates, were determined at a defined endpoint. A lag period was observed in which no tripalmitate was formed. The content of Lipozyme used was the most powerful factor in eliminating tripalmitate formation and reducing diglycerides; by using large quantities of Lipozyme, the reaction reached the endpoint before the tripalmitate formation became measurable and low levels of diglycerides were formed. The effects of varying the ratio of palmitic acid to triolein were investigated. A complex relationship between the ratio of substrate components emerged in which the diglyceride content increased with increasing triolein concentration and the tripalmitate content was lowest at a molar ratio of palmitic acid to triolein of 3.5. The reaction was run at 70, 80, and 90°C; best results were obtained at 70°. The water activity of the reaction was adjusted prior to catalysis and maintained during the reaction by equilibrating the reaction mixture and enzyme and running the reaction in an atmosphere of controlled water activity. A direct relationship between diglycerides and water activity was observed, and the level of tripalmitate formed corresponded to the time required to reach the endpoint. The reaction system was tested using ethyl palmitate instead of palmitic acid as acyl donor; the diglyceride content again increased with increasing water activity, but larger amounts of diglycerides were formed. Much shorter reaction times were required, with small quantities of tripalmitate formed.     

Molecular species analysis of 1,2-diglycerides stimulated by alpha-thrombin in cultured fibroblasts

Diglycerides derived from the phospholipase C-mediated hydrolysis of phosphoinositides are implicated as important mediators of agonist-induced responses, including the stimulation of cell division. alpha-Thrombin-stimulated proliferation of fibroblasts is associated with a sustained increase in cellular diglycerides, while the hydrolysis of phosphoinositides is transient (Wright, T. M., Rangan, L. A., Shin, H. S., and Raben, D. M. (1988) J. Biol. Chem. 263, 9374-9380). A rigorous assessment of this apparent discrepancy requires an analysis of the molecular species of the lipids involved. In this report, we have analyzed the molecular species of 1,2-diglycerides present in quiescent and alpha-thrombin-stimulated IIC9 Chinese hamster embryo fibroblasts. The molecular species profiles of the stimulated diglycerides were compared to the profiles of molecular species contained in cellular phospholipids. We demonstrate that 1) stimulation of IIC9 cells by alpha-thrombin results in an increase in the levels of diglyceride molecular species already present in control, quiescent cultures, without the addition of new species or the complete loss of existing species; 2) the diglycerides present in control cultures as well as in cultures stimulated with alpha-thrombin are all ester-linked; and 3) while the phosphoinositides contribute a significant proportion of the diglycerides generated 15 s following alpha-thrombin addition, phosphatidylcholine contributes most of the diglycerides generated after 5 min and 1 h.     

Incorporation of palmitic acid-1-14C into lipids of pharate adult tissues of Bombyx mori

Incorporation of palmitic acid-1-¹⁴C into pharate adult tissues and their lipid components of Bombyx mori was investigated. Rapid incorporation of radioactivity took place predominantly in fat body and haemolymph lipids, and partially in ovarian lipids immediately after the injection at the middle stage of pharate adult development. The major parts of the radioactivities in fat body, haemolymph and ovary were distributed in triglycerides and phospholipids, diglycerides, and triglycerides, respectively. The patterns of time course of incorporation of radioactivity into lipid components of pharate adult tissues suggest that the major form of lipid released from fat body may be diglycerides and the diglycerides in haemolymph are probably the main source of ovarian triglycerides.     

Detection and isolation of minor lipid constituents

The use of thick layers of adsorbent for the concentration and subsequent isolation of neutral lipid constituents is described. Lipids not perceptible by conventional methods are demonstrated in concentrates of bovine heart extracts and identified as fatty aldehydes, O-alk-1-enyl diglycerides (neutral plasmalogens), and O-alkyl diglycerides (alkoxydiglycerides).     

Non-ideal phase behaviour of binary mixtures of triglycerides with mono- and diglycerides

Non-ideal miscibility in the liquid phase of mono- or diglycerides with triglycerides has been described in terms of thermodynamic excess functions. To this end the heat of mixing and the solubility temperatures of several ideal and non-ideal binary glyceride mixtures have been measured. The non-ideal phase behaviour of glyceride systems reported in the literature has been satisfactorily predicted, which indicates that the excess functions are generally applicable to mixtures of mono- or diglycerides with triglycerides.     

Manipulation of galactolipid Fatty Acid composition with substituted pyridazinones

The fatty acid composition of the major lipids of the chloroplast membranes, the mono- and digalactosyl diglycerides, can be definably altered with various substituted pyridazinones. Galactolipid fatty acid composition of wheat (Triticum aestivum L.) can be altered so that there is a decrease in linolenic acid accompanied by an increase in linoleic acid without a shift in the relative proportion of saturated to unsaturated fatty acids; the fatty acid composition can be shifted toward a higher proportion of saturated fatty acids; or the fatty acid composition of the monogalactosyl diglycerides can be altered in preference to the digalactosyl diglycerides. Also, the light-mediated parallel accumulation of chlorophyll and linolenic acid can be separated with a substituted pyridazinone. The substituted pyridazinones may be useful tools in clarifying the role the galactolipids and their component fatty acids play in the structure and function of chloroplast membranes in higher plants.     

Physiological studies on Phymatotrichum omnivorum VI. Lipid composition of mycelium and sclerotia

The lipid composition of the mycelium and sclerotia ofPhymatotrichum omnivorum was compared. The lipids of the mycelium contained 47.9 % polar lipids as compared to 21.4 % in the sclerotia. Sterols represented 10 % of the lipids in sclerotia as contrasted to 3.6 % of the mycelium. More monoglycerides (17.5 %) were detected in the sclerotia as compared to the mycelium (1.6 %). Fatty acid analysis indicated that linoleic acid was the predominant fatty acid in the total fatty acids fraction in both the mycelium and the sclerotia. Palmitic acid was the major free fatty acid in the mycelium, whereas myristic acid was the predominant free fatty acid in the sclerotia. In the fatty acids of the diglycerides of sclerotia, palmitic acid represented 71 % of that fraction, as compared to 6.6 % of the fatty acids of the diglycerides in the mycelium. The major fatty acid in the diglycerides of the mycelium was oleic acid.     

Effects of mezerein and diglycerides on the PRL stimulation of cell replication in Nb2 node lymphoma cells

These studies provide further support for the thesis that the activation of protein kinase C is likely involved in the prolactin (PRL) stimulation of mitogenesis in the Nb2 node lymphoma cell line. The diterpene mezerein is shown to potentiate the mitogenic effect of PRL at a hormone concentration which elicits a less than maximum response. A similar response was observed with two diglycerides, diolein and dicaprin. Neither mezerein nor the diglycerides affected the magnitude of response to a maximum stimulatory concentration of PRL.     

Galactolipids and phospholipids of orange peel and juice chromoplasts

Chromoplasts from yellow orange (Citrus sinensis) fruit peel contain monogalactosyl diglycerides (MGDG), digalactosyl diglycerides (DGDG) and phosphatidyl glycerol (PG) in amounts similar to those found in chloroplasts from green fruit peel. Juice chromoplasts contain relatively little MGDG and no DGDG with high levels of phosphatidyl choline and phosphatidyl ethanolamine but no PG.     

The incorporation of [14C]acetate into the constituent fatty acids of monogalactosyldiglyceride by isolated spinach chloroplasts

The incorporation into diglycerides of the acyl products synthesized from acetate by spinach chloroplasts was greatly stimulated by the addition of glycerol 3-phosphate. When UDP-galactose was added also, monogalactosyldiglycerides became the major products. Palmitate biosynthesis was stimulated about twofold by these additions, while oleate biosynthesis decreased slightly, so that oleate:palmitate ratios were in the range 0.6 to 0.8 rather than about 1.6 when glycerol 3-phosphate and UDP-galactose were not added. On the other hand, Triton X-100 greatly stimulated both oleate and palmitate biosynthesis to give oleate:palmitate ratios of about 2.0. The proportions of oleate and palmitate in the newly synthesized diglycerides, or in monogalactosyldiglycerides when exogenous UDP-galactose was added, did not always reflect the proportions of these two fatty acids synthesized from acetate. When oleate:palmitate ratios were ?1, equal amounts were incorporated into diglycerides or into monogalactosyldiglycerides. When oleate:palmitate ratios were <1, incorporation of palmitate into diglycerides and monogalactosyldiglycerides exceeded that of oleate. 1-Oleoyl, 2-palmitoyl glycerol compounds were the principal products under all conditions but 1,2-dipalmitoyl compounds were also quantitatively important when glycerol 3-phosphate alone, or glycerol 3-phosphate together with UDP-galactose, was added. The distribution of label in the constituent glycerol and fatty acid moieties when monogalactosyldiglycerides were synthesized from diglycerides is consistent with galactosylation occurring without modification or exchange of fatty acids. The distribution of 16- and 18-carbon acyl residues between the 1 and 2 stereospecific positions of newly synthesized monogalactosyldiglyceride was typical of the endogenous polyene monogalactosyldiglycerides. However when palmitate synthesis was in excess of oleate synthesis some palmitate was esterified in position 1, whereas in the endogenous monogalactosyldiglycerides hexadecatrienoate is confined to position 2.     

Separation of mono- and diglycerides by gas--liquid chromatography

The parameters affecting the separation and quantification of trimethylsilyl ethers of mono- and diglycerides have been investigated by gas-liquid chromatography with QF-1 and SE-30 as stationary phases and a flame ionization detector. Results have been compared with those obtained earlier for triglycerides. The isothermal characteristics of a range of trimethylsilyl ethers of mono- and diglycerides on both stationary phases showed that log retention volume was directly proportional to carbon number and inversely proportional to absolute temperature. However, glyceride derivatives with lower carbon numbers deviated from these relationships. By using various rates of programmed temperature rise, we have determined the elution temperatures (Kelvin scale) of the mono- and diglyceride trimethylsilyl ethers relative to that of glycerol trilaurate. The "carbon equivalent of a trimethylsilyl group" is defined and shown to be useful in comparing the chromatographic properties of different glyceride classes. Weight and molar correction factors have been obtained and used to analyze diglycerides derived from egg and bovine brain lecithins.     

Synthesis and anti-herpes simplex viral activity of monoglycosyl diglycerides

Based on the discovery of antiviral beta-galactosyl diglycerides from Clinacanthus nutans leaves, 19 monoglycosyl diglycerides were synthesized and examined for inhibitory effect on herpes simplex virus types 1 and 2 (HSV-1, HSV-2). A study of the structure-activity relationships of the synthetic monoglycosyl diglycerides indicated that the fatty acyl moieties were critical for inhibitory action with higher activity displayed as the acyl groups became more olefinic in character. The sugar moiety was also important for anti-HSV action; however, the type of sugar (glucose or galactose) did not affect activity. The stereochemistry at C-2 of the glycerol backbone displayed no significant effect on anti-HSV activity. Among the compounds synthesized, 1,2-O-dilinolenoyl-3-O-beta-D-glucopyranosyl-sn-glycerol showed the highest inhibitory activity against HSV-1 and HSV-2 with IC50 values of 12.5+/-0.5 and 18.5+/-1.5 microg/ml, respectively.     

Genomic library screens for genes involved in n-butanol tolerance in Escherichia coli

Background

n-Butanol is a promising emerging biofuel, and recent metabolic engineering efforts have demonstrated the use of several microbial hosts for its production. However, most organisms have very low tolerance to n-butanol (up to 2% (v/v)), limiting the economic viability of this biofuel. The rational engineering of more robust n-butanol production hosts relies upon understanding the mechanisms involved in tolerance. However, the existing knowledge of genes involved in n-butanol tolerance is limited. The goal of this study is therefore to identify E. coli genes that are involved in n-butanol tolerance.

Methodology/Principal Findings

Using a genomic library enrichment strategy, we identified approximately 270 genes that were enriched or depleted in n-butanol challenge. The effects of these candidate genes on n-butanol tolerance were experimentally determined using overexpression or deletion libraries. Among the 55 enriched genes tested, 11 were experimentally shown to confer enhanced tolerance to n-butanol when overexpressed compared to the wild-type. Among the 84 depleted genes tested, three conferred increased n-butanol resistance when deleted. The overexpressed genes that conferred the largest increase in n-butanol tolerance were related to iron transport and metabolism, entC and feoA, which increased the n-butanol tolerance by 32.8±4.0% and 49.1±3.3%, respectively. The deleted gene that resulted in the largest increase in resistance to n-butanol was astE, which enhanced n-butanol tolerance by 48.7±6.3%.

Conclusions/Significance

We identified and experimentally verified 14 genes that decreased the inhibitory effect of n-butanol tolerance on E. coli. From the data, we were able to expand the current knowledge on the genes involved in n-butanol tolerance; the results suggest that an increased iron transport and metabolism and decreased acid resistance may enhance n-butanol tolerance. The genes and mechanisms identified in this study will be helpful in the rational engineering of more robust biofuel producers.