Excess Of Glycerol Research Articles

Expression of a Rhizopus oryzae lipase in Pichia pastoris under control of the nitrogen source-regulated formaldehyde dehydrogenase promoter

A Rhizopus oryzae lipase gene has been expressed in Pichia pastoris as a reporter using the formaldehyde dehydrogenase 1 promoter ( PFLD1) of this organism, which has been reported to be strongly and independently induced by either methanol as sole carbon source or methylamine as sole nitrogen source. Levels of lipase expressed and secreted under the control of the PFLD1 at different induction conditions have been compared to those obtained with the commonly used alcohol oxidase 1 promoter ( PAOX1) in small (shake flask) and 1 l bioreactor batch cultures. PFLD1-controlled heterologous gene expression was strongly repressed by excess of either glycerol or glucose–but not sorbitol–during growth using methylamine both as sole nitrogen source and inducing substrate. Co-induction of PFLD1 with methanol and methylamine resulted in a synergistic effect on extracellular lipase expression levels. In all tested conditions, the substitution of ammonium for methylamine as carbon source provoked a clear decrease in the specific growth rate and yield of biomass per gram of carbon source. Overall, this study demonstrates that the PFLD1 promoter is at least as efficient as the PAOX1 for extracellular expression of heterologous proteins in P. pastoris bioreactor cultures and provides a first basis for the further design of methanol-free high cell density fed-batch cultivation strategies for controlled overproduction of foreign proteins in P. pastoris.

Polymer supported nitrite esters as diazotising reagent

Summary Glycidyl methacrylate based terpolymer resin was prepared in spherical beads form by suspension polymerization of glycidyl methacrylate (0.4mol)-methyl methylacrylate (0.5mol)-divinyl benzene (0.1mol) mixture. Acid catalysed reaction of the resin beads (110-220 µm) with excess of glycerol gives corresponding modified resin with hydroxyl functions (6.86 mmol g -1 ). Upon standing in 2 M HNO 2 solution, hydroxyl groups of the later esterified with nitrous acid (3.55 mmol g -1 ). The nitrite ester functions of the resin have been demonstrated to be efficient nitrous acid precurser in diazotisation of aromatic amines. Diazotisation of aniline in 2 M HC1 solutions at O°C takes place in less than 15 minutes. Applicability and pratical yields of the method presented have been investigated by diazo coupling reaction of s- naphthol.

Reaction of glycerol with fatty acids in the presence of ion-exchange resins: Preparation of monoglycerides

The preparation of monoglycerides from glycerol and fatty acids in the presence of ion-exchange resins is studied. A preliminary swelling in the glycerol inhibits the reaction, whereas a swelling of the resin in the oleic acid, before the reaction, increases the activity and the selectivity. This result shows that the competitive adsorption of glycerol and fatty acid influences greatly the esterification rate. Generally, water is an inhibitor of the esterification reaction (catalyst poisoning and thermodynamic equilibrium) and different procedures have already been suggested for its elimination. By using a molecular sieve as a water trap, the oleic acid conversion increases up to 90% without any change of the monoglyceride selectivity (85%). Moreover, owing to the pore structure of the catalyst, it has to be expected that the hydrocarbon chain length of the fatty acid will influence the activity and the selectivity, evidence of which we give in the present study. Finally, if an excess of glycerol increases the selectivity of the monoglycerides, it decreases the activity and can be considered as a selective inhibitor.

Reactions of olive oil and glycerol over immobilized lipases

AbstractThe reaction of olive oil and glycerol over immobilized lipases was studied. For oil samples with free fatty acid (FFA) contents larger than 2%, FFA esterification and glycerolysis took place simultaneously, but the esterification reaction was faster than glycerolysis. Similar product distributions were obtained for glycerol/oil mole ratios of 3:1 and 6:1. Therefore, an excess of glycerol does not result in a significant increase in monoglyceride yield within the experimental range tested. The main reaction product at 80°C was diglyceride. No increase in monoglyceride yield was observed by lowering the reaction temperature to 10°C.

Papain catalyzed synthesis of glyceryl esters ofN-protected amino acids and peptides for the use in trypsin catalyzed peptide synthesis

Papain catalyzed synthesis of glyceryl esters of BOC(Z)-protected amino acids and peptides was performed at 40-50 degrees C in a 50 molar excess of glycerol. Equilibrium was achieved in 6-7 h. The maximal yield of esters (50-70%) was obtained at 10% of water and pH 3.2-3.4. A lower water concentration resulted in a sharp decrease of the ester yield. The synthesized glyceryl esters of neutral amino acids are good substrates for trypsin and can be used for peptide synthesis catalyzed for trypsin. Glycerol esters are also good substrates for other enzymes possessing esterase activity.

Papain catalyzed synthesis of glyceryl esters of N‐protected amino acids and peptides for the use in trypsin catalyzed peptide synthesis

Papain catalyzed synthesis of glyceryl esters of BOC(Z)-protected amino acids and peptides was performed at 40–50°C in a 50 molar excess of glycerol. Equilibrium was achieved in 6–7 h. The maximal yield of esters (50–70%) was obtained at 10% of water and pH 3.2–3.4. A lower water concentration resulted in a sharp decrease of the ester yield. The synthesized glyceryl esters of neutral amino acids are good substrates for trypsin and can be used for peptide synthesis catalyzed for trypsin. Glycerol esters are also good substrates for other enzymes possessing esterase activity. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 287–290, 1997.

Oligomers of a polyester network

AbstractOligomers of glycerol and succinic acid have been prepared by three different methods and characterised by solution 13C‐NMR spectroscopy at 125.8 MHz. The first series of five oligomers was prepared by reacting glycerol with succinic anhydride; only one acid function of each succinic acid residue formed an ester by this means. They were readily distinguished as the shifts of their glycerol residues were dispersed over 16 ppm, and their shifts provided a guide to the assignment of shifts in the more elaborate oligomers. The second set of oligomers was prepared by treating glycerol with a small quantity of succinic acid, ester links being promoted by means of the reagent dicyclohexylcarbodiimide (DCCDI). When we used 2 mol of DCCDI/mol of acid in the presence of an excess of glycerol, no free acid functions remained, and a new set of oligomers was obtained. Furthermore, within this set the proportion of ring molecules was enhanced by repeating the reaction under much more dilute conditions. In this way the shifts of two ring oligomers were recognized in the spectrum. A different set of oligomers again was obtained when the esterification was performed with 1 mol of DCCDI/mol of succininic acid. After the first generation of oligomers had been identified in the mixtures produced by these three experiments, the reactions continued to produce larger oligomers with new fine structure features in the spectra. Glycerol trisuccinate was prepared in a pure form and heated in a vacuum to eliminate succinic acid groups and allow the formation of oligomers with two and three branch points. The structures successively produced by this reaction were readily recognized. In all we were able to recognize the formation of component structures in at least 17 different oligomers. The shifts of the carbons of the glycerol residues are sensitive to the substitution pattern at that residue, to whether the succinic acid residue to which they are linked has reacted a second time, and in some cases the methine shift is sensitive to how the succinic acid residue attached to a neighboring methylene carbon has reacted. © 1994 John Wiley & Sons, Inc.

Use of lipases in multiphasic systems solely composed of substrates

Three lipase‐catalyzed reactions have been investigated in relation to specificity and water dependence. The reactions in question include: the synthetic reaction between oleic acid and glycerol; the enzymatic hydrolysis of triolein; and alcoholysis/glycerolysis transesterification reactions. All reactions were carried out under solventfree conditions. In each case, the medium composition and reaction conditions were optimized in order to work at elevated substrate concentrations and to minimize the production of by‐products. Different lipase preparations have been tested in each reaction. In the synthetic reaction, the effective removal of produced water was found to be vital for the production of triolein. With water removal and glycerol amounts not higher than required by the stoichiometry of the reaction, 95% of the available oleic acid was converted to triolein in 48 hr. The production of triolein was also found to be dependent on the availability of the 1,2‐diglyceride to react with oleic acid. In the hydrolysis reaction, best conversion yields of triolein towards monoolein, diolein and free fatty acid were obtained when water was considered simply as a substrate of the reaction. In glycerolysis reactions, the reaction of triolein to give monoolein and diolein followed much the same pattern as for hydrolysis, when water was replaced by glycerol. It was shown again that near stoichiometric amounts of substrates led to the best conversion to mono‐ and diglycerides. A small excess of glycerol was found to be very inhibitory to the reaction. All possible isomers were formed during the reaction. Conversely, in alcoholysis reactions between triolein and stearyl alcohol the specificity of the lipase was upheld. Excess alcohol in this instance was found to be beneficial.

Glycerolysis of fats and methyl esters — Status, review and critique

AbstractWith the possible exception of catalytic hydrogenation, perhaps no unit operation within the realm of oleochemistry is as thoroughly complex as that of glycerolysis. Among the misconceptions and half‐truths that prevail concerning the glycerolysis of fats are the notions that it involves a strictly random distribution of acyl groups among all of the available hydroxyl groups, that the solubility of glycerol in the fat at the reaction temperature determines the yield of monoglyceride that may be obtained, that the advantageous effects of the Law of Mass Action can be realized only when the reaction media is in ultimate homogeneity, in other words, complete mutual solubility, and, more importantly, that there is an equivalence of emulsification properties for the chief products of glycerolysis, i.e., the α‐and β‐monoglycerides, in both food and industrial emulsification. Numerous examples from international literature establish the limitations which prevail in temperature, agitation and use of excess of glycerol in batch glycerolysis reactions, but the practical limits for glycerolysis undersuperemulsification conditions remain to be established. The disadvantages of glycerolysis in homogeneous solvents still are insufficient to justify the use of those that are available, but the use of both pressure and gaseous catalysts such as carbon dioxide appear to offer the greatest hope for improvement in yields. Substantial energy savings may dictate the choice of methyl ester glycerolysis processing for future plants, especially those in the international sphere. Pros and cons of monoglyceride analytical methodology are evaluated.

Conditions for sulfite stabilization and determination by ion chromatography

The efficiencies of three groups of potential sulfite-stabilizing compounds were found to be in the order: carbonyls > alcohols = saccharides. A mole ratio of 1:1 between formaldehyde and sulfite was sufficient for stabilizing a sulfite solution for at least 72 h. The lower stabilizing efficiencies of the alcohols and saccharides examined could be compensated by using large excesses of these compounds. For example, if a 100-fold excess of glycerol over sulfite was used, the recovery of sulfite was 96% after 72 h compared with only 40% without addition of stabilizer. During separations by ion chromatography, almost no oxidation of the sample occurs provided the sample solution is directly injected into a deaerated eluent. For formaldehyde, the peak heights were found to depend on the molar ratio of the stabilizer to sulfite as well as on the concentration of sulfite. This effect was not found for the other stabilizers tested.

Reactions and Reaction Products on the Synthesis of Monooleoylglycerol

Investigations on the direct esterification of oleic acid and equimolecular or a large excess of glycerol were carried out under the conditions of reaction temperatures at 125280°C, for 36h in the absence of a catalyst. Reaction products were determined by the TLS.1) The optimum preparative conditions for maximum monooleoylglycerol were given and under these, quantitative results of the product were obtained as follows : Molar ratio of oleic acid and glycerol, 1 : 5, Reaction temperature and time, 250°C, 3h, Weight ratio of component, monooleoylglycerol (MO) 78% (1-MO 52%, 2-MO 26%), dioleoylglycerol (DO) 21% (1, 3-DO 1.3%, 1, 2-DO 8%), trioleoylglycerol and oleic acid, trace.2) It was recognized that reactioas of oleic acid (O) with glycerol (G) could be separated into three types : (a) Esterification (molar ratio of O/G=1/1, at 125200°C) (b) Reactions among acylglycerols formed by esterification (O/G=1/1, approximately at 250°C) (c) Glycerolysis (O/G=1/3, 1/5, approximately at 250°C)

Lipid Metabolism in Cultured Cells: XI. UTILIZATION OF SERUM TRIGLYCERIDES

Utilization of serum triglycerides by cells in tissue culture has been studied in order to determine their importance as a source of cell lipids and to obtain information on the cellular mechanisms of triglyceride uptake. L strain mouse fibroblasts incorporated serum triglycerides from the growth medium, but the rates of uptake were up to 10-fold less than that of free fatty acids or monoglycerides under the same culture conditions. When cells were grown under conditions of limited supply of serum lipids, however, only about 7% of the cell lipid came from serum free fatty acids and up to 28% was provided by the serum triglycerides. The major portion of the cell lipid under these conditions was derived by de novo synthesis from glucose. Hydrolysis of exogenous triglyceride by lipases in the serum-supplemented tissue culture medium was measured and found to be inadequate to account for the rate of triglyceride utilization observed in these studies. In addition, heat treatment of the serum to inactivate lipase did not significantly affect triglyceride uptake. No excretion of lipase activity by the cells was detected. Incorporation of triglyceride labeled in the glycerol portion with 3H and the fatty acid portion with 14C was measured from untreated, heat-inactivated, or delipidized serum using both monolayers and growing cultures. The 3H:14C ratio in the isolated cellular lipids averaged over 70% of that in the original triglyceride under all conditions of culture. Furthermore, incorporation of the [3H]glycerol portion of the molecule was not diluted out by addition to the growth medium of a large excess of nonradioactive glycerol or a nonhydrolyzable monoglyceride analog. In short term uptake experiments most of the radioactivity in cell lipids was found in the triglyceride fraction, with only minor amounts in the free fatty acids. In long term growth experiments the incorporated triglyceride was converted extensively (70 to 80%) to cellular phospholipid with maintenance of the 3H:14C ratio. The results indicate that L cells in tissue culture can utilize serum triglycerides and suggest that the predominant mechanism of uptake involves the intact molecule and does not require prior hydrolysis.

D-mannosaminuronic acid constituent of the K-7 antigen of Escherichia coli

The identification of d-mannosaminuronic acid (2-amino-2-deoxy-d-mannuronic acid) as one of the main constituents of an acidic polysaccharide material isolated from two Escherichia coli serotypes is described. This material carrying the specificity of the E. coli K7 antigen is isolated from serotypes O14:K7:H− and O7:K7:H4 by extraction of the bacterial dry mass–obtained by growth on solid medium–using the hot phenol-water procedure. Fractionation of the aqueous phase material, after removal of lipopolysaccharide by repeated ultracentrifugation, was achieved by a cetavlon precipitation technique. RNA, representing the bulk of the aqueous phase material, precipitates in the presence of cetavlon already at a NaCl molarity of 0.3 M whereas the K7 active material is only precipitable at a much lower NaCl molarity of 0.06. Examination of HCl-hydrolysates of this substance by high voltage electrophoresis in a non-complexing buffer of pH 5.3 revealed the presence of a positively charged component which was shown to be the lactone of an aminohexuronic acid. The amino sugars present in the K7 specific material were separated from uncharged hydrolysis products by passing the hydrolysate over a Dowex 50 column previously equilibrated with 0.1 N HCl. The amino sugar fraction obtained by elution with 0.5 N HCl consisted of three major components: (a) a hexosaminuronic acid, (b) the lactone of the hexosaminuronic acid and (c) d-glucosamine. (a) and (b) are interconvertible and give rise to the same product after N-acetylation. (a) Shows the characteristic ninhydrin staining of hexosaminuronic acids and does react in the Elson-Morgan reaction with a maximal absorption at 530–533 mμ, thus indicating a free 1-aldo-2-aminogroup and, in addition, an unsubstituted C-3 position. Chromatographic and electrophoretic data differed from those of two hexosaminuronic acid standards available (glucosaminuronic and galactosaminuronic acids) but were in agreement with data reported by Perkins [2] for mannosaminuronic acid isolated from a polysaccharide material of Micrococcus lysodeikticus. Since no reference material was available we tried to elucidate the structure of the hexosaminuronic acid by converting the previously esterified hexosaminuronic acid units of the polymer by NaBH4-reduction to the related hexos-amine units. Esterification was carried out with an aqueous solution of ethylene oxide at room temperature, and NaBH4-reduction in the presence of an excess of glycerol in order to maintain a pH of approximately 8. Examination of the reduced material revealed the presence of a newly formed hexosamine which subsequently was identified as d-mannosamine. The identification is based on the following results: a) identity of Rf- and MGlcn-values in several systems of the free and the N-acetylated amino sugar with authentic samples of d-mannosamine and N-acetyl-d-mannosamine; b) formation of a pentose with the Rf-value of arabinose after ninhydrin degradation; c) reactivity of the amino sugar with hexokinase and formation of a hexosamine-6-phosphate. This reaction in addition proves the d-configuration of the amino sugar. Quantitative analysis shows that d-mannosaminuronic acid forms at least 15–20% of the K7 specific material extracted from E. coli O14:K7, whereas d-glucosamine represents only a minor constituent (2–3%). Hitherto 2-amino-2-deoxy-mannuronic acid was only reported once [2] as a constituent of a cell wall polysaccharide from Micrococcus lysodeikticus. To our knowledge its occurence in the K7 specific material of E. coli is the first report from gramnegative bacteria.

Studies of the Cross-linking and Glass Transition of Fatty Acid-Modified Alkyd Resins

Abstract a) The degree of cross-linking observed in films of fatty acid modified alkyd resins increases with the content of unsaturated acids. Moreover, cross-linking is not so effective in raising the Tg as the benzene ring, which has a larger cohesive energy. b) Both the Tg and the degree of cross-linking increase with the increase in the molecular weight of the prepolymer. c) The excess of glycerol employed in fatty acid-modified alkyd resin is effective in raising the Tg and encouraging the progress of the cross-linking. d) Generally, the cross-linking is not soon completed; as time elapses, the Tg rises, and the cross-linking progresses. No change in the Gh with the elevation of the Tg (baked film with the addition of a drier) is considered to be caused by the formation of polar groups by air-oxidation. e) In the early period of drying, a sub-glass transition caused by the unrestrained chain of the acyl groups is observed.

A monoglyceride‐rich tung oil product

AbstractA method for preparing tung monoglycerides (1) by the reaction of tung oil with an excess of glycerol in the presence of sodium methoxide as the catalyst has been adapted to the preparation of large batches of material. Laboratory scale studies have shown that a product analyzing as containing up to 50% of tung monoglycerides is produced when the reaction is carried out at 150ŶC. for one hour with an excess of 1 mole of glycerol and 1% of sodium methoxide (based on oil weight). The data were applied successfully to three batchwise pilotplant preparations using from 30 to 47 pounds of tung oil to produce a product containing from 32% to 45% tung monoglycerides. Unreated glycerol was successfully separated from the product by centrifugation, with no water or salt solution washing, to give a product containing 1.5% to 3.5% free glycerol.Difficulties were encountered in analyzing the product for monoglyceride, and it was found that the triene conjugation contributed to high results, However, there also appear to be components other than simple monoglycerides in the product which analyze as monoglycerides to give a falsely high value, as evidenced by differences found by analyses of the products before and after essentially complete hydrogenation.

The preparation and purification of monoglycerides

AbstractMono‐ and diglycerides were produced by reacting the following oils with glycerol: coconut, peanut, sesame, linseed, and sardine. It was shown that the yield of monoglyceride was not dependent upon the fatty acid composition of the oil but on the solubility of glycerol in oil, which is dependent in part on the temperature. An excess of glycerol above that which is soluble does not change the composition of the reaction product. At 180°C, no more than 45% monoglycerides can be formed by glycerolysis.

抗痙攣薬の合成研究 (第5報)

Myanesin was successfully obtained by allowing calcium monoglyceroxide to absorb carbon dioxide in an anhydrous state, in the presence of excess of glycerol and o-cresol, with subsequent heating to a high temperature. This method of preparation is more simple of procedures and more economical than the existing general method starting with glycerol α-monochlorhydrin.