Epoxidation of unsaturated fatty acids by a soluble cytochrome P-450-dependent system from Bacillus megaterium

In previous publications from this laboratory we have described a soluble, partially purified cytochrome P-450-dependent monooxygenase complex that, in the presence of NADPH and O2, catalyzes the monohydroxylation of long chain fatty acids, alcohols, and amides at the omega -1, omega -2, and omega -3 positions. We have now found that this preparation catalyzes the epoxidation as well as the hydroxylation of palmitoleic acid and a variety of other monounsaturated fatty acids. The experimental results reported here strongly support the concept that both hydroxylation and epoxidation are catalyzed by an identical cytochrome P-450 complex utilizing the same active and binding sites. Furthermore, for saturating levels of these substrates, the rate-limiting step in oxygenation does not appear to involve substrate structure. Thus, although the position and geometry of the double bond may dramatically affect the rate of epoxidation relative to hydroxylation, the combined rate of substrate oxygenation is essentially a constant independent of this ratio. Finally, we propose and present evidence for an enzyme-substrate binding model that involves polar binding of the carboxyl terminus and strong hydrophobic binding and sequestering of the terminal methyl group of the fatty acid. The three methylene carbons adjacent to the methyl group are positioned in a set geometry around the active site but the midchain region of a monounsaturated fatty acid is relatively free to interact or bind loosely with the enzyme surface in a variety of conformations. Depending on fatty acid structure, one or more of these conformations can bring the unsaturated center close enough to the active site to permit epoxidation of the double bond.     

Microbial production of a novel trihydroxy unsaturated fatty acid from linoleic acid

A bacterium isolated from a dry soil sample collected from McCalla, AL, USA, converted linoleic acid to a novel compound, 12,13,17-trihydroxy-9 (Z)-octadecenoic acid (THOA). The organism is a Gram-positive, non-motile rod (0.5 μ m × 2 μ m). It was identified as a species of Clavibacter ALA2. The product was purified by high pressure liquid chromatography, and its structure was determined by ¹H and ¹³C nuclear magnetic resonance and Fourier transform infrared spectroscopies, and by mass spectrometer. Maximum production of THOA with 25% conversion of the substrate was reached after 5–6 days of reaction. THOA was not further metabolized by strain ALA2. This is the first report of a 12,13,17-trihydroxy unsaturated fatty acid and its production by microbial transformation. Some dihydroxy intermediates were also detected. THOA has a structure similar to those of known plant self-defense substances. Received 13 January 1997/ Accepted in revised form 05 May 1997     

The protonophore resistance of Bacillus megaterium is correlated with elevated ratios of saturated to unsaturated fatty acids in membrane phospholipids

Growth of the protonophore-resistant strain of Bacillus megaterium, strain C8, in the presence of oleic acid markedly reduced its resistance to low concentrations of carbonylcyanide m-chlorophenylhydrazone (CCCP). Growth of the CCCP-sensitive wild-type strain in the presence of stearic acid increased the resistance of that strain to growth inhibition by protonophore. Studies of the membrane lipids indicated that in the absence of additions to the medium, membranes from C8 contained greatly reduced levels of monounsaturated fatty acids relative to the wild type; wild-type levels were restored by growth of C8 in the presence of oleic acid, concomitant with the loss of resistance. Conversely, growth of the wild type on stearic acid increased the ratio of saturated/unsaturated fatty acids in the membrane, concomitant with a modest increase in the resistance of the wild-type strain to CCCP. The exogenous oleic acid was preferentially incorporated into phosphatidylethanolamine, diphosphatidylglycerol, and 1,2-diacylglycerol, whereas stearic acid was incorporated preferentially into phosphatidylglycerol, and into the small component of free fatty acids. Depending upon the growth conditions, changes in membrane lipid-to-membrane protein ratio and in the ratios of polar lipid components were observed, but none of those changes correlated as did the changes in saturated fatty-acid-to-unsaturated fatty-acid ratio with protonophore resistance. This latter correlation was further suggested by experiments in which the protonophore resistance of wild type B. megaterium was shown to increase with increasing growth temperature without any temperature-dependent loss of protonophore efficacy. The experiments here support the hypothesis developed from work with Bacillus subtilis that changes in the fatty acid composition of the membrane phospholipids affect energy coupling, and make it clear that simple increases or decreases in the hydrolytic activity of ATPase in the uncoupler-resistant mutants of bacilli are not correlated with resistance in some direct way.     

Regulation of the synthesis of unsaturated fatty acids by growth temperature in Bacillus subtilis

Bacillus subtilis synthesizes, almost exclusively, saturated fatty acids, when grown at 37° C. When cultures were transferred from 37° C to 20° C, a chloramphenicol- and rifampicin-sensitive synthesis of a C-16 unsaturated fatty acid was observed. Synthesis of this compound reached a plateau after 5 h at 20° C, reaching levels of 20% of the total fatty acid content. [¹⁴C]-labelled fatty acids attached as thioesters to acyl-carriers compounds, such as coenzyme A (CoA) or acyl-carrier protein (ACP) synthesized de novo by glycerol-requiring auxotrophs deprived of glycerol to arrest phospholipid synthesis, could not be desaturated at 20° C. Desaturation of these fatty acids was readily observed when glycerol was restored to the cultures allowing resumption of transfer of acyl-moieties from acyl-thioesters to phospholipid. It was also observed that depletion of the pools of CoA and ACP by starvation of pantothenate auxotrophs had no effect on the observed synthesis of unsaturated fatty acid at 20° C. The overall results indicate that synthesis of unsaturated fatty acids in B. subtilis is a cold-inducible process and that phospholipids are obligate intermediates in this fatty acid desaturation pathway.     

不饱和脂肪酸在逆境胁迫中的作用

生物膜是将细胞与环境分开的第一道屏障,是环境胁迫造成损伤的主要位点.脂肪酸是生物膜的主要组成成分,不饱和脂肪酸在决定生物膜的生理特性中具有重要作用,增加脂肪酸的不饱和程度能增加膜脂的流动性.近年来,很多研究发现,生物通过脂肪酸脱饱和维持膜的流动性来适应外界环境变化.本文主要从不饱和脂肪酸在环境温度胁迫、盐胁迫、氧化胁迫、酸碱胁迫、干旱胁迫、乙醇胁迫及铝胁迫中的作用研究进展进行了综述.     

Production of novel tetrahydroxyfuranyl fatty acids from alpha-linolenic acid by Clavibacter sp. strain ALA2

Previously, it was reported that a newly isolated microbial culture, Clavibacter sp. strain ALA2, produced trihydroxy unsaturated fatty acids, diepxoy bicyclic fatty acids, and tetrahydroxyfuranyl fatty acids (THFAs) from linoleic acid (C. T. Hou, J. Am. Oil Chem. Soc. 73:1359-1362, 1996; C. T. Hou and R. J. Forman III, J. Ind. Microbiol. Biotechnol. 24:275-276, 2000; C. T. Hou, H. Gardner, and W. Brown, J. Am. Oil Chem. Soc. 75:1483-1487, 1998; C. T. Hou, H. W. Gardner, and W. Brown, J. Am. Oil Chem. Soc. 78:1167-1169, 2001). In this study, we found that Clavibacter sp. strain ALA2 produced novel THFAs, including 13,16-dihydroxy-12-THFA, 15-epoxy-9(Z)-octadecenoic acid (13,16-dihydroxy-THFA), and 7,13,16-trihydroxy-12, 15-epoxy-9(Z)-octadecenoic acid (7,13,16-trihydroxy-THFA), from alpha-linolenic acid (9,12,15-octadecatrienoic acid). The chemical structures of these products were determined by gas chromatography-mass spectrometry and proton and (13)C nuclear magnetic resonance analyses. The optimum incubation temperature was 30 degrees C for production of both hydroxy-THFAs. 13,16-Dihydroxy-THFA was detected after 2 days of incubation, and the concentration reached 45 mg/50 ml after 7 days of incubation; 7,13,16-trihydroxy-THFA was not detected after 2 days of incubation, but the concentration reached 9 mg/50 ml after 7 days of incubation. The total yield of both 13,16-dihydroxy-THFA and 7,13,16-trihydroxy-THFA was 67% (wt/wt) after 7 days of incubation at 30 degrees C and 200 rpm. In previous studies, it was reported that Clavibacter sp. strain ALA2 oxidized the C-7, C-12, C-13, C-16, and C-17 positions of linoleic acid (n-6) into hydroxy groups. In this case, the bond between the C-16 and C-17 carbon atoms is saturated. In alpha-linolenic acid (n-3), however, the bond between the C-16 and C-17 carbon atoms is unsaturated. It seems that enzymes of strain ALA2 oxidized the C-12-C-13 and C-16-C-17 double bonds into dihydroxy groups first and then converted them to hydroxy-THFAs.     

Studies on the production of conjugated linoleic acid from linoleic and vaccenic acids by mixed rumen protozoa

The conversion of β-myrcene to the furanoid flavour compound perillene by Pleurotus ostreatus was investigated using trideutero β-myrcene, trideutero α-(Z)-acaridiol and non-labeled 1,2- and 3,10-epoxy-β-myrcene, α,α-acarilactol, and perillene as substrates. Myrcene diols were formed from the cleavage of myrcene epoxides, but only α-(Z)-acaridiol, a 1,4-butanediol derivative most likely generated through a base-catalysed epoxide opening, was a suitable precursor of perillene. Once formed, this key intermediate was rapidly oxidised and the resulting cyclic lactol was dehydrated to yield perillene. Bioconversion of the supplemented perillene to α,α-acariolide indicated that perillene was another intermediate of the pathway and prone to further oxidative degradation. The data suggest that the fungus converted the cytotoxic β-myrcene in its environment into a metabolically useable carbon source along this route.     

Production of dicarboxylic acids from novel unsaturated fatty acids by laccase-catalyzed oxidative cleavage

The establishment of renewable biofuel and chemical production is desirable because of global warming and the exhaustion of petroleum reserves. Sebacic acid (decanedioic acid), the material of 6,10-nylon, is produced from ricinoleic acid, a carbon-neutral material, but the process is not eco-friendly because of its energy requirements. Laccase-catalyzing oxidative cleavage of fatty acid was applied to the production of dicarboxylic acids using hydroxy and oxo fatty acids involved in the saturation metabolism of unsaturated fatty acids in Lactobacillus plantarum as substrates. Hydroxy or oxo fatty acids with a functional group near the carbon–carbon double bond were cleaved at the carbon–carbon double bond, hydroxy group, or carbonyl group by laccase and transformed into dicarboxylic acids. After 8 h, 0.58 mM of sebacic acid was produced from 1.6 mM of 10-oxo-cis-12,cis-15-octadecadienoic acid (αKetoA) with a conversion rate of 35% (mol/mol). This laccase-catalyzed enzymatic process is a promising method to produce dicarboxylic acids from biomass-derived fatty acids.     

Effect of unsaturated fatty acids and Ca2+ on phosphatidylinositol synthesis and breakdown

CDP-diglyceride : inositol transferase was inhibited by unsaturated fatty acids. The inhibitory activity decreased in the following order: arachidonic acid greater than linolenic acid greater than linoleic acid greater than oleic acid greater than or equal to palmitoleic acid. Saturated fatty acids such as myristic acid, palmitic acid, and stearic acid had no effect. Calcium ion also inhibited the activity of CDP-diglyceride : inositol transferase. In rat hepatocytes, arachidonic acid inhibited 32P incorporation into phosphatidylinositol and phosphatidic acid without any significant effect on 32P incorporation into phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine. Ca2+ ionophore A23187 also inhibited 32P incorporation into phosphatidylinositol. However, 32P incorporation into phosphatidic acid was stimulated with Ca2+ ionophore A23187. Phosphatidylinositol-specific phospholipase C was activated by unsaturated fatty acids. Polyunsaturated fatty acids such as arachidonic acid and linolenic acid had a stronger effect than di- and monounsaturated fatty acids. Saturated fatty acids had no effect on the phospholipase C activity. The phospholipase C required Ca2+ for activity. Arachidonic acid and Ca2+ had synergistic effects. These results suggest the reciprocal regulation of phosphatidylinositol synthesis and breakdown by unsaturated fatty acids and Ca2+.     

Synthesis of hydroxy fatty acids from linoleic acid by human blood platelets

The metabolism of linoleic acid by washed human platelets was investigated. [1.14C] linoleic acid was converted to [1.14C] hydroxy octadecadienoic acids (HODEs) at about the same rate with which [1.14C] 12-HETE was produced from [1.14C] arachidonic acid. The total radioactivity in HODEs was distributed among two isomers: 13-HODE (85%) and 9-HODE (15%) as defined by CG-MS. The production of HODEs by intact washed platelets was inhibited by indomethacin (IC50:5 x 10(-7) M) which suggest that hydroxy fatty acids were produced by PGH-synthase. By contrast, the production of HODEs by platelet cytosolic fractions was not modified under indomethacin treatment but completely abolished by NDGA (10(-3) M) and inhibited by the platelet lipoxygenase inhibitors 15-HETE (2.10(-5) M) and baicalein (10(-5) M). Platelets thus contain two different active systems which may convert linoleic acid to hydroxy fatty acids. Since these compounds remained essentially associated with the platelets, their presence may significantly participate in the mechanisms of platelet activation.     

Synthesis of hydroxy fatty acids from linoleic acid by human blood platelets

The metabolism of linoleic acid by washed human platelets was investigated. 1.¹⁴C linoleic acid was converted to 1.¹⁴C hydroxy octadecadienoic acids (HODES) at about the same rate with which 1.¹⁴C 12-HETE was produced from 1.¹⁴C arachidonic acid. The total radioactivity in HODEs was distributed among two isomers: 13-HODE (85%) and 9-HODE (15%) as defined by GC-MS. The production of HODES by intact washed platelets was inhibited by indomethacin (IC50:5×10⁻⁷M) which suggest that hydroxy fatty acids were produced by PGH-synthase. By contrast, the production of HODEs by platelet cytosolic fractions was not modified under indomethacin treatment but completely abolished by NDGA (10⁻³M) and inhibited by the platelet lipoxygenase inhibitors 15-HETE (2.10⁻⁵M) and baicalein (10⁻⁵M). Platelets thus contain two different active systems which may convert linoleic acid to hydroxy fatty acids. Since these compounds remained essentially associated with the platelets, their presence may significantly participate in the mechanisms of platelet activation.     

Reaction conditions for maximal cyclodextrin production by cyclodextrin glucanotransferase from Bacillus megaterium

The effect of the reaction conditions (substrate concentration, enzyme dosage, and pH) on cyclodextrin production by cyclodextrin glucanotransferase from Bacillus megaterium was investigated by applying mathematical modeling methods. Adequate models were developed and they were used for determination of the optimal conditions for maximal formation of beta-cyclodextrins at minimal concentrations of a- and gamma-cydclodextrins. The main factor affecting the ratio of the products was pH of the reaction mixture. At pH 9 the enzyme formed mainly beta- and y-cyclodextrins and the ratio a:beta:gamma was 2.6:83.5:13.9; at pH 5 the ratio changed to 8.6:84.6:6.8. Mathematical models were used for determination of the conditions for maximal conversion of the substrate into cyclodextrins. 45.88% conversion of starch was achieved at 5% substrate concentration, 3.5 U/g enzyme dosage, and pH 7.4.