Production of 5,8,11-cis-eicosatrienoic acid by a Δ12-desaturase-defective mutant ofMortierella alpina 1S-4

A mutant defective in Δ12-desaturase of an arachidonic-acid producing fungus,Mortierella alpina 1S-4, was shown to be a novel potent producer of Mead acid (5,8,11-cis-eicosatrienoic acid, 20:3ω9). The fungus produced several fatty acids of the n-9 family,i.e., 6,9-cis-octadecadienoic acid (18:2ω9), 8,11-cis-eicosadienoic acid (20:2ω9) and 20:3ω9. Significantly high levels of these fatty acids were produced during growth at low temperatures (12–20°C). On submerged cultivation at 20°C for 10 days in a 5-L fermenter containing 2% glucose plus 1% yeast extract (pH 6.0), the production of 20:3ω9 reachedca. 0.8 g/L (56 mg/g dry mycelia), accounting for 15% (by wt) of the total mycelial fatty acids. The other major fatty acids were palmitic acid (6%), stearic acid (11%), oleic acid (45%), 18:2ω9 (12%) and 20:2ω9 (3%). Studies on the distribution of fatty acids among lipid classes showed that, irrespective of the growth temperature employed (12–28°C),ca. 70% (by mol) of 20:3ω9 was present in the triglyceride and the remainder in the phospholipid fraction, especially in phosphatidylcholine (PC). When the fungus was grown at 12°C, the proportion of 20:3ω9 in the PC fraction wasca. 55%. On leave from Suntory Ltd.     

Synthesis of dodecanedioic acid fromvernonia galamensis oil

Dodecanedioic acid, currently available from elaborate synthesis utilizing petrochemical feedstock, was synthesized fromVernonia galamensis oil via an efficient, relatively simple three-reaction sequence that involved the room temperature oxidation of 12,13-epoxystearic acid. The first and second steps of the reaction scheme were saponification and hydrogenation, both of which were almost quantitative. The third reaction, involving chromic acid oxidation of the resulting epoxy acid, afforded greater than 50% of the theoretical yield of dodecanedioic acid (>95% purity, m.p. 123–125°C; lit. m.p. 128–130°C).     

Utilization of high-melting palm stearin in lipase-catalyzed interesterification with liquid oils

Palm stearin with a melting point (m.p.) of 49.8°C was fractionated from acetone to produce a low-melting palm stearin (m.p.=35°C) and a higher-melting palm stearin (HMPS, m.p.=58°C) fraction. HMPS was modified by interesterification with 60% (by weight) of individual liquid oils from sunflower, soybean, and rice bran by means of Mucor miehei lipase. The interesterified products were evaluated for m.p., solid fat content, and carbon number glyceride composition. When HMPS was interesterified individually with sunflower, soybean or rice bran at the 60% level, the m.p. of the interesterified products were 37.5, 38.9, and 39.6°C, respectively. The solid fat content of the interesterified products were 30–35 at 10°C, 17–19 at 20°C, and 6–10 at 30°C, respectively. The carbon number glyceride compositions also changed significantly. C₄₈ and C₅₄ glycerides decreased remarkably with a corresponding increase of the C₅₀ and C₅₂ glycerides. All these interesterified products were suitable for use as trans acid-free and polyunsaturated fatty acid-rich shortening and margarine fat bases.     

Conversion of linoleic acid to 10-hydroxy-12(Z)-octadecenoic acid byFlavobacterium sp. (NRRL B-14859)

A new microbial isolate,Flavobacterium sp. strain DS5, converts linoleic acid into 10-hydroxy-12(Z)-octadecenoic acid (10-HOA) with 55% yield. The product was characterized by gas chromatography (GC), GC/mass spectrometry, nuclear magnetic resonance and Fourier transform infrared spectroscopy. The specific optical rotation of 10-HOA is [α] _D ²⁴ =−5.58 (methanol). The optimum time, pH and temperature for the production of 10-HOA were 36h, 7.5 and 20–35°C, respectively. The enzyme(s) that converts linoleic acid to 10-HOA is soluble and located intracellulary in strain DS5. Two minor products, 10-methoxy-12-octade-cenoic acid and 10-keto-12-octadecenoic acid, were also identified. 10-HOA was further metabolized by strain DS5. Among the unsaturated fatty acids studied, the order of reactivity for the DS5 enzyme(s) is oleic>palmitoleic> linoleic>linolenic>γ-linolenic>myristoleic acid.     

Increase in the γ-linolenic acid content by solvent winterization of fungal oil extracted fromMortierella genus

The fungal oil extracted fromMortierella ramanniana var.angulispora (IFO 8187) was solvent winterized in order to raise the content of γ-linolenic acid (GLA). Effects of winterization conditions (solvent, oil concentration in the solvent and temperature) and changes of glyceride compositions were discussed. The fungal oil was separated into four diglycerides and 17 triglycerides (TG) with high performance liquid chromatography. The predominant species were POO, POP and LOP, whose contents were 24.4, 22.9 and 9.4% of the total TG, respectively. Ethanol at 4°C gave the highest GLA content of 10.5% in spite of lower yield than with acetone at −20°C. The highest separation efficiency for GLA (η_GLA) was 0.27 with acetone at −20°C and 10% oil concentration, resulting in 8.3% of GLA from the fungal oil at 5.7% LGA. In case of lower oil concentration at 5–20%, η_GLA showed higher in the following order: acetone (−20°C)>n-hexane (−20°C)>acetone (4°C)>petroleum ether (−20°C). The winterization process also proved to be effective for the separation of TG type, Sa₂U (Sa; saturated fatty acid; U, unsaturated fatty acid) into the crystallized fraction and SaU₂ into the liquid fraction. Acetone at −20°C showed higher separation efficiency for triunsaturated TG than the other solvents.     

Biocatalytic production of 10-hydroxystearic acid, 10-ketostearic acid, and their primary fatty amides

The objective of this study was to develop scaleup bioprocesses for producing 10-hydroxystearic acid (10-HSA) and 10-ketostearic acid (10-KSA) as well as their primary amides for potential new uses. A reactor process was examined to obtain the mono-oxygenated FA using Sphingobacterium thalpophilum (NRRL B-14797) and Bacillus sphaericus (NRRL NRS-732), which solely produce 10-HSA and 10-KSA, respectively, from technical-grade oleic acid. By using an 8-h-old B-14797 culture grown in a manganese-containing WF6 medium, pH 7.3, at 28°C under 350 rpm agitation and 0–50% dissolved oxygen concentrations provided by a controlled sparger aeration, the production of 10-HSA reached 7 g/L with a 40% yield in 4 d. In using a 12-h-old NRS-732 culture grown in a pyruvate-containing PF6 medium, pH 6.5, at 30°C under 750 rpm agitation without any sparger aeration during the conversion reaction, 10-KSA production reached 7.9 g/L with a yield of more than 54% in 72 h. The scaleup reactor process provided crystalline 10-HSA and 10-KSA for producting new primary amides via a lipase-catalyzed amidation reaction with yields of 94 and 92%, respectively. The primary amides of 10-HSA and 10-KSA displayed m.p of 115 and 120°C, respectively.     

Purification and characterization of thermophilic and alkalophilic tributyrin esterase fromBacillus strain A30-1 (ATCC 53841)

An extracellular esterase (EC 3.1.1.1) from a thermophilicBacillus A30-1 (ATCC 53841) was purified 139-fold to homogeneity by sodium chloride (6 M) treatment, ammonium sulfate fractionation (30–80%) and phenyl-Sepharose CL-6B column chromatography. The native enzyme was a single polypeptide chain with a molecular weight of about 65,000 and an isoelectric point at pH 4.8. The optimum pH for esterase activity was 9.0, and its pH stability range was 5.0–10.5. The optimum temperature for its activity was 60°C. The esterase had a half-life of 28 h at 50°C, 20 h at 60°C and 16 h at 65°C. It showed the highest activity on tributyrin, with little or no activity toward long-chain (12–20 carbon) fatty acid esters. The enzyme displayed K_m and K_cat values of 0.357 mM and 8365/min, respectively, for tributyrin hydrolysis at pH 9.0 and 60°C. Cyclodextrin (α, β, and γ), Ca²⁺, Co²⁺, Mg²⁺ and Mn²⁺ enhanced the esterase activity, and Zn²⁺ and Fe²⁺ acted as inhibitors of the enzyme activity. The enzyme activity was not affected by ethylenediaminetetraacetic acid, p-chloromercuribenzoate andN-bromosuccinimide. This paper was presented in part at the 82nd Annual Meeting and Exposition of the American Oil Chemists’ Society, held May 12–15, 1991, in Chicago, Illinois.     

Variation in fatty acid content and seed weight in some lauric acid richCuphea species

Significant genetic variation for lauric acid (12∶0) and capric acid (10∶0) composition and seed weight was measured within lauric acid-rich, self-pollinating germplasm accessions ofCuphea wrightii, C. tolucana, andC. lutea. Means and ranges of individual plant progenies for 12∶0 content ofC. wrightii accessions was 60.5±.63% (49.8±65.8%), 10∶0 content was 23.7±.54% (18.6±33.0%), and 1000-seed weight was 1.50±.03 g (1.20–2.47 g). Progenies of single plant selections carried to the S₂ generation exhibited reduced variability within selections, but significant variation among selections for 12∶0, 10∶0 and 1000-seed weight. Variation among single plant selections ofC. tolucana was less than that ofC. wrightii and attributed to a restricted germplasm base. Means and ranges for 12∶0 content were 61.6±.47% (59.2–69.9%), 10∶0 was 22.3±.62% (11.7–25.3%), and 1000-seed weight was 1.40±.05 g (0.90–1.69 g).Cuphea lutea has a significantly different 12∶0−10∶0 profile than the other lauric acid-rich species. Means and ranges for 12∶0 were 36.8±.14% (33.7–40.8%), 10∶0 was 21.8±.08% (16.4–23.9%), 1000-seed weight was 2.26±.02 g (1.82–272 g). The 1000-seed weight was highly positively correlated with 8∶0, 10∶0, 18∶1 and 18∶2 contents and highly negatively correlated with 12∶0, 14∶0 and 16∶0 in bothC. wrightii andC. tolucana. No such relationship was found forC. lutea. A highly significant negative correlation was also measured for 12∶0 and 10∶0 contents inC. wrightii andC. tolucana.     

The Di-t-butylation of p-cresol with t-butanol in Supercritical CO2 over Tungstophosphoric Acid Supported on Ordered Mesoporous Silica

Tungstophosphoric acid (HPW) supported on MCM-41 was an excellent catalyst for the t-butylation of p-cresol to 2,6-di-t-butyl-4-methylphenol (2,6-DTBPC) in supercritical CO₂; however, zeolites, H-Y and H-Beta, only gave 2-t-butyl-4-methylphenol (2-TBPC) because of their limitation in pore size. The yield of 2,6-DTBPC was maximized at 110 °C, and further increase in temperature rather decreased the yield. The yield of 2,6-DTBPC was maximized at 10–11 MPa CO₂ pressure, and further increase of the pressure decreased in the conversion of phenol and the yield of 2,4-DTBC. The thermogravimetric analysis of used catalysts showed that the coke-formation was minimized in supercritical CO₂ compared to the other reaction media such as in liquid phase and in N₂ atmosphere.     

Microbial conversion of palmitoleic acid to 9,12-hexadecadienoic acid (16:2ω4) by Trichoderma sp. AM076

Trichoderma sp. AM076, isolated from a freshwater sample, was found to accumulate 9,12-cis-hexadecadienoic acid (16:2ω4), when grown with palmitoleic acid (16: 1ω7). Methyl myristate was the best carbon source for the conversion of palmitoleic acid to 16:2ω4. The mycelial 16:2ω4 content reached 17.4 mg/g dry mycelia (443 mg/L) when the fungus was grown in a medium that contained 2.0% methyl myristate, 1.5% yeast extract, and 2.0% methyl palmitoleate, pH 6.0, for 5 d at 28°C with shaking. In both nonpolar and polar lipids from the mycelia, 16:2ω4 was detected as one of the major fatty acids when 16:1ω7 was added. It is probable that 16:1ω7 is converted to 16:2ω4 through the Δ12 desaturation reaction.     

Production of 14-Oxo-<Emphasis Type="Italic">cis</Emphasis>-11-eicosenoic Acid from Lesquerolic Acid by <Emphasis Type="Italic">Sphingobacterium multivorum</Emphasis> NRRL B-23212

The objective of this study was to explore the extent of microbial conversion of lesquerolic acid (14-hydroxy-cis-11-eicosenoic acid; LQA) by whole cell catalysis and to identify the newly converted products. Among compost isolates including NRRL strains B-23212 (Sphingobacterium multivorum), B-23213 (Acinetobacter sp.), B-23257 (Enterobacter cloacae B), B-23259 (Escherichia sp.) and B-23260 (Pseudomonas aeruginosa) the S. multivorum strain was the only microorganism that converted LQA to produce a new product identified as 14-oxo-cis-11-eicosenoic acid by GC-MS and NMR analyses. The conversion yield was 47.4% in 48 h at 200 rpm and 28°C in small shake flask experiments. In comparison, both Acinetobacter and Pseudomonas strains failed to convert LQA to major new products but used LQA apparently as an energy source during fermentation. For structural analysis, 6.88 g of 14-oxo-cis-11-eicosenoic acid was produced from converting 11 g LQA (a 62% yield) in 72 h at 200 rpm and 28 °C in Fernbach flasks using 18-h-old NRRL B-23212 cultures and an improved medium that also contained EDTA and glycerol in lieu of glucose as carbon source. NRRL B-23212 was further identified by 16S rRNA gene sequence analysis as a unique strain of S. multivorum. Therefore, S. multivorum NRRL B-23212 possesses an enzymatic activity presumably a secondary alcohol dehydrogenase for converting LQA to produce 14-oxo-cis-11-eicosenoic acid, a first report that demonstrates the functional modification of LQA by whole cell catalysis.     

Lipase-catalyzed synthesis of hydroxy stearates and their properties

Hydroxy fatty acid (HFA) esters of long-chain alcohols, such as hydroxy stearates, have potential applications from lubricants to cosmetics. These esters were synthesized enzymatically to overcome the problems associated with chemical processes. An immobilized lipase, Rhizomucor miehei, was employed as catalyst in the esterification reaction between hydroxy-stearic acid as a source of HFA and monohydric fatty alcohols (C₈–C₁₈). The yields of esters were in the range of 82–90% by conducting the reactions at 65±2°C, 2–5 mm Hg pressure, and 10% lipase concentration. The products were analyzed by infrared spectroscopy, and some of their analytical characteristics were determined.     

n‐octane reforming over alumina‐supported Pt, Pt–Sn and Pt–W catalysts

Pt, Pt–Sn and Pt–W supported on γ‐Al₂O₃ were prepared and characterized by H₂ chemisorption, TEM, TPR, test reactions of n‐C₈ reforming (500°C), cyclohexane dehydrogenation (315°C) and n‐C₅ isomerization (500°C), and TPO of the used catalysts. Pt is completely reduced to Pt⁰, but only a small fraction of Sn and of W oxides are reduced to metal. The second element decreases the metallic properties of Pt (H₂ chemisorption and dehydrogenation activity) but increases dehydrocyclization and stability. In spite of the large decrease in dehydrogenation activity of Pt in the bimetallics, the metallic function is not the controlling function of the bifunctional mechanisms of dehydrocyclization. Pt–Sn/Al₂O₃ is the best catalyst with the highest acid to metallic functions ratio (due to its lower metallic activity) presenting a xylenes distribution different from the other catalysts. The acid function of Pt–Sn/Al₂O₃ is tuned in order to increase isomerization and cyclization and to decrease cracking, as compared to Pt and Pt–W. This revised version was published online in July 2006 with corrections to the Cover Date.     

Differential Scanning Calorimetry Analysis of Goat Fats: Comparison of Chemical Composition and Thermal Properties

The physical–chemical properties, fatty acid composition and thermal properties of goat subcutaneous (SF), tallow (TF) and intestinal (IF) fats were determined. SF differed from other fat types with respect to its lower melting (41.6 °C), lower saponification (190.3 mg KOH/g) and higher iodine (40.4) values as compared to those of other fats. Goat fat types contained palmitic acid (C_16:0), stearic acid (C_18:0), oleic acid (C_18:1ω9) and linoleic acid (C_18:2ω6) as the major components of the fatty acid composition (23.06–23.52, 22.95–39.03, 21.94–36.16 and 1.96–2.22%, respectively). A differential scanning calorimetry (DSC) study revealed that two characteristic peaks were detected in both crystallization and melting curves. Major peaks (T _peak) of TF and IF were similar and determined as 34.02–35.24 and 9.95–10.72 °C, respectively for the crystallization peaks and 15.11–18.26 and 50.70–52.76 °C, respectively for the melting peaks in the DSC curves; but those of SF (27.14 and 4.36 °C for crystallization peaks and 8.39 and 44.93 °C for melting peaks) differed remarkably from those of other fat types.     

An investigation of the oil of laemonema morosum matsubara

Summary It has been shown that the oil ofLaemonema Morosum Matsubara, a deep sea fish, contains a large amount (31–34%) of unsaponifiable matter. The main component (ca. 50%) of which is 11-docosen-1-ol, has an m.p. 31.7–32.3°C. This alcohol has not been previously reported in the literature. From this alcoholtrans-11-docosen-1-ol having m.p. 52.4–52.8°C. was prepared. The infrared spectra of these two alcohols are given.     

Characterization and Immobilization of Marine Algal 11-Lipoxygenase from Ulva fasciata

The lipoxygenase (LOX) of the marine green alga Ulva fasciata was purified and immobilized in order to improve the stability and reusability. The algal LOX was partially purified by fractionation with 35–55% saturation of ammonium sulfate and MacroPrep high Q anion exchange chromatography. The LOX was purified ten times using linoleic acid (C_18:2) or arachidonic acid (C_20:4) as substrate, the Michaelis constant (K _m) of LOX was 117.6, 31.3 μM, and maximum velocity (V _max) was 12.8, 23.3 μmol hydroperoxy fatty acid/min-mg protein, respectively. The algal LOX showed the highest activity towards C_18:4 followed by C_20:4, C_18:2 and methyl ester of C_18:4. LOX activity increased up to 10.5 times with increased concentration of Triton X-100 in the extraction medium reaching an optimum at 0.05%. Calcium chloride, glutathione and phenylmethylsulphonyl fluoride were found effective protectants to LOX during purification. Hydroperoxyeicosatetraenoic acid (HpETE) formed from arachidonic acid catalyzed by this purified algal LOX was reduced and identified as 11-hydroxy-5,8,12,14-eicosatetraenoic acid (11-HETE) by NP-HPLC and GC–MS. This algal 11-LOX was immobilized in alginate beads. The stability was sevenfold greater than that of the unbound lipoxygenase at 4 °C in 0.05 M Tris–HCl buffer (pH 7.5). This is the first report on immobilization of a marine algal lipoxygenase with a view to its potential role in seafood flavor formation.     

Low-temperature selective catalytic reduction of NO with NH3 based on MnOx-CeOx/ACFN

MnO _x -CeO _x /ACFN were prepared by the impregnation method and used as catalyst for selective catalytic reduction of NO with NH3 at 80°C-150°C. The catalyst was characterized by N₂-BET, scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FT-IR). The fraction of the mesopore and the oxygen functional groups on the surface of activated carbon fiber (ACF) increased after the treatment with nitric acid, which was favorable to improve the catalytic activities of MnO _x -CeO _x /ACFN. The experimental results show that the conversion of NO is nearly 100% in the range 100°C-150°C under the optimal preparation conditions of MnO _x -CeO _x /ACFN. In addition, the effects of a series of performance parameters, including initial NH₃ concentration, NO concentration and O₂ concentration, on the conversion of NO were studied. __________ Translated from Chemical Industry and Engineering Progress, 2007, 27(1): 87–91 [译自: 化工进展]     

Four-factor response surface optimization of the enzymatic modification of triolein to structured lipids

The ability of an immobilized lipase to modify the fatty acid composition of (88.8% C_18:1, 4.3% C_16:0, 3.1% C_18:0, and 3.8% C_18:2 as determined by gas chromatography, and approximately 90% triolein) in hexane by incorporation of a medium-chain fatty acid, capric acid (C₁₀), to form structured triacylglycerol was studied. Response surface methodology was used to evaluate the effect of synthesis variables, such as reaction time (12–36 h), temperature (25–65°C), molar substrate ratio of capric acid to triolein (2:1–6:1), and enzyme amount (10–30% wt% of triacylglycerol), on the yield of structured lipid. Optimization of the transesterification was attempted to obtain maximum yield of structured lipid while using the minimum molar substrate ratio and enzyme amount as much as possible. Computer-generated contour plot interpretation revealed that a relatively high molar substrate ratio (6:1) combined with low enzyme amount (10%) after 30 h of reaction at 25°C gave optimum incorporation of capric acid. A total yield for combined monoand dicaproolein of up to 100% was obtained.     

Production of structured lipids by lipase-catalyzed acidolysis in supercritical carbon dioxide: Effect on acyl migration

Structured lipids were synthesized by the acidolysis of corn oil by caprylic acid in supercritical carbon dioxide (SCCO₂) with Lipozyme RM IM from Rhizomucor miehei. The effects of pressure and temperature on the reaction were studied. To compare the degrees of acyl migration in the SCCO₂ and solvent-free reaction systems, the effects of reaction time on the degree of acyl migration were also studied. The highest mole percentage incorporation of caprylic acid (62.2 mol%) occurred at 24.13 MPa in SCCO₂. The overall incorporation of caprylic acid in the SCCO₂ system remained higher than that in the solvent-free system at every temperature tested. This trend was observed more clearly at lower temperatures (35–55°C) than at higher temperatures (65–75°C). Acyl migration with both reaction systems was low, with a negligible difference between them up to 12 h, after which the degree of acyl migration in the solvent-free system increased rapidly with time up to 24 h compared with the SCCO₂ system.     

Oxidation of cyclopentene catalyzed by tungsten-substituted molybdophosphoric acids

A series of Keggin type tungsten-substituted molybdophosphoric acids (H₃PMo_12−n W _n O₄₀·XH₂O) were synthesized and characterized by ICP-AES, FT-IR, TG-DSC, and XRD. The tungsten substitution extent significantly affected their catalytic activity in the oxidation of cyclopentene and the selectivity of the resultant products. The tungsten-substituted molybdophosphoric acids with tungsten substitution numbers in a range of 3–6.8 exhibited high catalytic activity in the oxidation of cyclopentene. After reaction for 8 h, the conversion of cyclopentene was up to 97%; the oxidation products mainly consisted of glutaraldehyde, cis-1,2-cyclopentanediol and trans-1,2-cyclopentanediol with the yields of ca. 23%, 27%, and 45%, respectively.