Production of biodiesel by immobilized Candida sp. lipase at high water content

A new process for enzymatic synthesis of biodiesel at high water content (10–20%) with 96% conversion by lipase from Candida sp. 99–125 was studied. The lipase, a no-position-specific lipase, was immobilized by a cheap cotton membrane and the membrane-immobilized lipase could be used at least six times with high conversion. The immobilized lipase could be used for different oil conversion and preferred unsaturated fatty acids such as oleic acid to staturated fatty acids such as palmitic acid. The changes in concentration of fatty acids, diglycerides, and methyl esters in the reaction were studied and a mechanism of synthesis of biodiesel was suggested: the triglycerides are first enzymatically hydrolyzed into fatty acids, and then these fatty acids are further converted into methyl esters.     

Structural characterization of gentiobiosyl diglycerides fromBacillus pumilus associated with ascidiaHalocynthia aurantium

A mixture of 1(3),2-di-O-acyl-3(1)-O-β-gentiobiosylglycerols was isolated from a sea isolate ofBacillus pumilus. The components of the mixture were structurally characterized by mass spectrometry and¹H and¹³C NMR spectroscopy data for the native compounds and their derivatives. The predominant component contains two C₁₅ acyl groups, while the second component contains C₁₅ and C₁₇ fatty acids. Six minor components differ in residues of fatty acids and/or their combinations. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 1, pp. 166–170, January, 2000.     

Structural characterization of gentiobiosyl diglycerides fromBacillus pumilus associated with ascidiaHalocynthia aurantium

A mixture of 1(3),2-di-O-acyl-3(1)-O-β-gentiobiosylglycerols was isolated from a sea isolate ofBacillus pumilus. The components of the mixture were structurally characterized by mass spectrometry and¹H and¹³C NMR spectroscopy data for the native compounds and their derivatives. The predominant component contains two C₁₅ acyl groups, while the second component contains C₁₅ and C₁₇ fatty acids. Six minor components differ in residues of fatty acids and/or their combinations. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 1, pp. 166–170, January, 2000.     

Characterization of ganoderma spore lipid by stable carbon isotope analysis: implications for authentication

The ratios of stable carbon isotopes (¹³C/¹²C) of ganoderma fruiting body, ganoderma spore, ganoderma spore lipid (GSL) and individual fatty acids in GSL were determined by gas chromatography–stable isotope ratio mass spectrometry and elemental analysis–stable isotope ratio mass spectrometry. These values fall into a range from −26.9 to −23.3‰, suggesting that the cut log as the Ganoderma-cultivated substrate in Fujian, China, may belong to C3 plants. Eighteen fatty acids were identified and their abundances measured by gas chromatography–mass spectrometry in the six GSL samples with C_16:0, C_18:0, C_18:1 and C_18:2 as major constituents, and C_16:1 is evidently enriched compared with the other edible vegetable oils. On the basis of the compositions of fatty acids and stable carbon isotopes in GSL, we have developed a novel method to detect the adulteration of GSL products with cheaper edible vegetable oils. An example of ideal blending between GSL and C4 or C3 vegetable oil is further provided to expound the discrimination procedures and corresponding sensitive indicators. Simultaneously, the carbon isotope fractionation in the biosynthesis of individual fatty acids was observed, revealing that the formation of C_18:0 from C_16:0 in ganodema spores had no conspicuous ¹³C enrichment of +0.4‰ for Ganoderma sinensis spore and +0.1‰ for G. lucidum spore; the desaturation of C_18:0 to C_18:1 resulted in a distinct ¹³C depletion of −1.4‰ for G. sinensis spore and −0.9‰ for G. lucidum spore; and the next desaturation from C_18:1 to C_18:2 displayed no evident ¹³C fractionation of −0.1‰ for G. sinensis spore and −0.2‰ for G. lucidum spore. Figure Ganoderma lucidum has been widely used in traditional Chinese medicines. Ganoderma spore lipid (GSL) extracted from the spores of G. lucidum has been approved as a health food supplement. However, because of rarity, GSL has become a target for adulteration with cheaper vegetable oils.     

Purification and Characterization of an Organic Solvent-Tolerant Lipase from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> CS-2

An extracellular lipase secreted by Pseudomonas aeruginosa CS-2 was purified to homogeneity about 25.5-fold with an overall yield of 45.5%. The molecular mass of the lipase was estimated to be 33.9 kDa by SDS-PAGE and 36 kDa by gel filtration. The optimum temperature and pH were 50 °C and 8.0. The lipase was found to be stable at pH 4–10 and below 50 °C. Its hydrolytic activity was highest against p-nitrophenyl palmitate (p-NPP) among p-nitrophenyl esters of fatty acids with various chain lengths. The lipase was activated in the presence of Ca²⁺, while it was inactivated by other metal ions more or less. EDTA significantly reduced the lipase activity, indicating the lipase was a metalloenzyme. Gum Arabic and polyvinyl alcohol 124 enhanced lipase activity but Tween-20, Tween-80, and hexadecyltrimethyl ammonium bromide strongly inhibited the lipase. It exhibited stability in some organic solvents. The lipase was activated in the presence of acetonitrile. Conversely, it was drastically inactivated by methanol and ethanol.     

A β-glucosidase from Sclerotinia sclerotiorum

The filamentous fungus Sclerotinia sclerotiorum, grown on a xylose medium, was found to excrete one β-glucosidase (β-glu x). The enzyme was purified to apparent homogeneity by ammonium sulfate precipitation, gel filtration, anion-exchange chromatography, and high-performance liquid chromatography (HPLC) gel filtration chromatography. Its molecular mass was estimated to be 130 kDa by HPLC gel filtration and 60 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, suggesting that β-glu x may be a homodimer. For p-nitrophenyl β-d-glucopyranoside hydrolysis, apparent K _m and V _max values were found to be 0.09 mM and 193 U/mg, respectively, while optimum temperature and pH were 55–60°C and pH 5.0, respectively. β-Glu x was strongly inhibited by Fe²⁺ and activated about 35% by Ca²⁺. β-Glu x possesses strong transglucosylation activity in comparison with commercially available β-glucosidases. The production rate of total glucooligosaccharides (GOSs) from 30% cellobiose at 50°C and pH 5.0 for 6 h with 0.6 U/mL of enzyme preparation was 80 g/L. It reached 105 g/L under the same conditions when using cellobiose at 350 g/L (1.023 M). Finally, GOS structure was determined by mass spectrometry and ¹³C nuclear magnetic resonance spectroscopy.     

Monolayer properties of fatty acids : II. Surface vapor pressure and the free energy of compression

The properties of monolayers spread at the air-water interface were measured for saturated, unsaturated, and hydroxy fatty acids, differing in the type and degree of unsaturation, geometric isomerism, and position of unsaturated and hydroxy groups. Surface vapor pressures, reflecting the equilibrium between “gaseous” and “liquid” monolayer states were determined, as were the free energies of compression, ΔF_c, from essentially infinite dilution (100,000 Ų/molecule) to the area per molecule, A_e at the equilibrium spreading pressure, π_e. Surface vapor pressures and free energies of compression for saturated and unsaturated fatty acids with a double bond, or bonds, change in a manner expected because of chain-chain interactions. Hydroxy and acetylenic acids produce relatively high surface vapor pressures, despite their tendency for strong chain-chain interaction. It is concluded that chain-water interactions are very significant for the acetylenic and hydroxy acids and less so for the saturated and ethylenic acids.     

RADICAL PROCESSES IN LIPIDS. A LASER PHOTOLYSIS STUDY OF t-BUTOXY RADICAL REACTIVITY TOWARD FATTY ACIDS

Laser photolysis techniques have been used to measure the reactivity of t-butoxy radical (t-BuO) toward various fatty acids. Eight compounds varying both in number and in the configuration of olefinic bonds were examined. It has been found that the rate constant for hydrogen abstraction from these compounds by t-BuO may be related to the number of secondary, allylic, and doubly allylic hydrogens in each molecule by the equation: k_r= [0.072[H_sec] + 0.518[H_allylic] + 2.716[H_{doubly allylic}] x 10⁶M^-1 s^-1.     

Significantly Improved Expression and Biochemical Properties of Recombinant Serratia marcescens Lipase as Robust Biocatalyst for Kinetic Resolution of Chiral Ester

A lipase gene from Serratia marcescens ECU1010 was cloned into expression vector pET28a, sequenced, and overexpressed as an N terminus His-tag fusion protein in Escherichia coli. Through the optimization of culture conditions in shake flask, the lipase activity was improved up to 1.09 × 10⁵ U/l, which is a great improvement compared to our previous reports. It was purified to homogeneity by Ni-NTA affinity chromatography with an overall yield of 59.4% and a purification factor of 2.4-fold. This recombinant lipase displayed excellent stability below 30 °C and within the pH range of 5.0−6.8, giving temperature and pH optima at 40 °C and pH 9.0, respectively. The lipase activity was found to increase in the presence of metal ions such as Ca²⁺, Cu²⁺, and some nonionic surfactants such as PEG series. In addition, among p-nitrophenyl esters of fatty acids with varied chain length, the recombinant lipase showed the maximum activity on p-nitrophenyl laurate (C₁₂). Using racemic trans-3-(4′-methoxy-phenyl)-glycidyl methyl ester [(±)-MPGM] as substrate, which is a key chiral synthon for production of diltiazem, a 50% conversion yield was achieved after 4 h in toluene–water (100 mM KPB phosphate buffer, pH 7.5) biphasic system (5:5 ml) at 30 °C under shaking condition (160 rpm), affording (−)-MPGM in nearly 100% ee. The K _m and V _max values of the lipase for (±)-MPGM were 222 mM and 1.24 mmol min⁻¹ mg⁻¹, respectively. The above-mentioned features make the highly enantioselective lipase from Serratia marcescens ECU1010 a robust biocatalyst for practical use in large-scale production of diltiazem intermediate.     

Starch Derivatives of High Degree of Functionalization 11: Studies on Alternative Acylation of Starch with Long-chain Fatty Acids Homogeneously in <Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-dimethyl acetamide/LiCl

Starch materials of different amylose content were allowed to react with saturated and unsaturated fatty acids of varying chain length from C₁₄ to C₁₈ under homogeneous conditions applying the solvent N,N-dimethyl acetamide in combination with LiCl. As reagent the corresponding acid chlorides and, alternatively, carboxylic acids in situ activated with toluene-4-sulfonyl chloride and N,N′-carbonyldiimidazole (CDI) were studied. Using fatty acid chlorides (FACl) and toluene-4-sulfonyl chloride-activated acids an almost complete substitution of the starch occurs. By in situ activation of the fatty acids with CDI, products with a degree of substitution up to 2 were obtained, which, in particular, represents a simple and very mild procedure.     

Purification and properties of uricase fromCandida sp. and its application in uric acid analysis in serum

The purification of uricase fromCandida sp. was carried out by precipitation with ammonium sulfate then further proceeded with Sephadex G200, and DEAE-cellulose DE52 chromatographies. The specific activity of the enzyme was enhanced from 0.05-12 (U/mg protein). The purity of the enzyme was judged to be homogeneous by SDS-PAGE. Some of the general properties of enzyme were investigated. The optimum reaction pH and temperature were 8.5 and 30‡C, respectively. The enzyme was stable at a pH range from 8.5-9.5 and at temperatures lower than 35‡C. The apparentK _m value of the enzyme was calculated to be about 5.26 x 10^-6 mol/L. The molecular weight was determined to be 70,000-76,000 by the gel filtration and SDS-PAGE techniques. The isoelectric point was determined to be pH 5.6. The effects of some metallic ions on enzyme activity and stability were discussed. The partial purified uricase was used in serum uric acid determination. The within-batch imprecision percentage ranged from 2.16-2.63 and the between-batch imprecision percentage ranged from 2.4-3.6. The recovery ratio were from 96–101%. The correlation among this method and Boehringer, Roche, or Biotrol enzymatic kits were Y = 1.086x - 0.50 (r = 0.981),Ya = 0.959x - 0.29 (r = 0.97), andYb = l.ll0x - 0.45(r = 0.956), respectively. A linear calibration curve was obtained at 2.5-15 mg/dL uric acid. The stability of reagents and the effects of some substances in serum were also surveyed.     

Purification and partial characterization ofRhizomucor miehei lipase for ester synthesis

A commercialRhizomucor miehei lipase was purified by ammonium sulfate precipitation. Phenyl Sepharose 6 Fast Row hydrophobic interaction chromatography, and DEAE Sepharose Fast Flow anion-exchange chromatography. The recovery of lipase activity was 32% with a 42-fold purification. The molecular size of the purified enzyme was 31,600 Dalton and the pI 3.8. The enzyme was stable for at least 24 h within a pH range of 7.0-10.0, and 96.8% of the enzyme activity remained when kept at 30‡C for 24 h. Further, about 10–30% of the lipase activity was inhibited by K⁺, Li⁺, Ni⁺, Co²⁺, Zn²⁺, Mg²⁺, Sn²⁺, Cu²⁺, Ba²⁺, Ca²⁺, and Fe²⁺ ions and by SDS, but EDTA had no effect. Under the experimental conditions, the optimum temperature for the hydrolysis of olive oil was 50‡C (pH 8.0), and for the synthesis of 1-butyl oleate, 37‡C. It was concluded that hydrolytic activity of lipase alone is not a sufficient criterion for its synthetic potential. The optimal molar ratio of oleic acid and 1-butanol was 2:1 for 1-butyl oleate synthesis. The 1-butyl oleate yield was unaffected by purification of the enzyme after 12 h.     

FT-midIR determination of fatty acid profiles, including trans fatty acids, in bakery products after focused microwave-assisted Soxhlet extraction

A study of the feasibility of Fourier transform medium infrared spectroscopy (FT-midIR) for analytical determination of fatty acid profiles, including trans fatty acids, is presented. The training and validation sets—75% (102 samples) and 25% (36 samples) of the samples once the spectral outliers have been removed—to develop FT-midIR general equations, were built with samples from 140 commercial and home-made bakery products. The concentration of the analytes in the samples used for this study is within the typical range found in these kinds of products. Both sets were independent; thus, the validation set was only used for testing the equations. The criterion used for the selection of the validation set was samples with the highest number of neighbours and the most separation between them (H<0.6). Partial least squares regression and cross validation were used for multivariate calibration. The FT-midIR method does not require post-extraction manipulation and gives information about the fatty acid profile in two min. The 14:0, 16:0, 18:0, 18:1 and 18:2 fatty acids can be determined with excellent precision and other fatty acids with good precision according to the Shenk criteria, R ²≥0.90, SEP=1–1.5 SEL and R ²=0.70-0.89, SEP=2–3 SEL, respectively. The results obtained with the proposed method were compared with those provided by the conventional method based on GC-MS. At 95% significance level, the differences between the values obtained for the different fatty acids were within the experimental error. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Effects of L-Carnitine and its Derivatives – Studies on Isolated Hearts

Summary. The metabolism in the heart prefers long-chain fatty acids to other substrates. L-Carnitine, a co-factor of coenzyme A, plays an essential role in the transport of long-chain fatty acids through the inner mitochondrial membrane. Without carnitine, metabolisation of long-chain fatty acids in the mitochondria is not possible. In addition, acyl groups from acyl-CoA compounds can be transferred to L-carnitine, thus influencing the enzymatic activities of important mitochondrial enzymes.The isolated heart model developed by Langendorff was used to investigate the effects of L-carnitine on the heart. During aerobic perfusion, the hemodynamic parameters of isolated hearts reacted in a very sensitive way to alterations in the external conditions (temperature, preload, composition of the perfusion solution). During postischemic perfusion, recovery of the hearts was also influenced by the composition of the perfusion. The hemodynamic parameters of the reperfused hearts increased markedly if there was a sufficiently high supply of long-chain fatty acids and/or glucose. The insufficient recovery of hearts perfused without glucose and at low fatty acid concentrations could be improved by adding L-carnitine. Determination of carnitine levels in heart tissue found that the heart loses about 30% of its carnitine content during ischemia, and that exogenous carnitine is taken up by the heart during reperfusion. There it effects the restoration of sufficient concentrations of creatine phosphate and ATP, a fact that was confirmed by ³¹P NMR spectroscopy. NMR spectroscopy also established that L-carnitine lessens the harmful effects of ischemia-induced metabolic acidosis.The favourable influence of L-carnitine on the heart in the reperfusion period could be due to a reduction in oxygen radicals (lowering of MDA concentrations during reperfusion, raising of GP_x and SOD activities).The findings of these experiments on isolated hearts as well as the favourable results of two placebo-controlled and double-blind clinical studies (investigating the effects of carnitine in cardiomyopathy patients and the effects of L-carnitine in hemodialysis patients) demonstrate that L-carnitine produces positive therapeutic effects, particularly in heart and circulatory diseases.     

Synthesis of Chiral 12-Phenyl(2H)dodecanoic Acids: Useful metabolic probes for the biosynthesis of 1-alkenes from fatty acids

The synthesis of chiral 12-phenyi(²H)dodecanoic acids as metabolic probes for the evaluation of the stereo-chemical course of the biosynthesis of 1-alkerses from fatty acids in plants and insects is described. The diastereoisomeric (2R, 3R)- or (2S, 3S)-12-phenyl(2,3?²H₂)dodecanoic acids 11 are obtained in high chemical and optical yield (>97% e.e.) from the readily available (E)-12-phenyl(2,3-²H₂)dodec-2-enoic acid ( 10 ) or (E)-12-phenyldodec-2-enoic acid ( 10a ) by microbial reduction with wet packed cells of Clostridium tyrobutyricum in either ²H₂O or H₂O buffer. (2R)- and (2S)-12-phenyl(2?²H)dodecanoic acids 9 (>97% e.e.) are accessible from the allylic alcohol 6 via Sharpless epoxidation with (+)-L- or (?)-D-diethyl tartrate, Synthetic routes to the (E)- and (Z)-11-phenyl(1?²H) undec-1-enes 16 and 16a as reference compounds are also included.     

Determination of 30 Free Fatty Acids in Two Famous Tibetan Medicines by HPLC with Fluorescence Detection and Mass Spectrometric Identification

A simple and sensitive high-performance liquid chromatographic (HPLC) method with fluorescence detection and mass spectrometric identification has been developed for analysis of 30 long-chain and short-chain free fatty acids (FFAs). The fatty acids were derivatized to their esters with 1-[2-(p-toluenesulfonate)ethyl]-2-phenylimidazole-[4,5-f]-9,10-phenanthrene (TSPP) in N,N-dimethylformamide (DMF) at 90 °C with anhydrous K₂CO₃ as catalyst. A mixture of C₁–C₃₀ fatty acids was completely separated within 60 min by gradient elution on a reversed-phase C₈ column. Qualitative identification of the acids was performed by atmospheric-pressure chemical ionization mass spectrometry (APCI–MS) in positive-ion mode. The fluorescence excitation and emission wavelengths were 260 and 380 nm, respectively. Quantitative determination of the 30 acids in two Tibetan medicines Gentiana straminea and G. dahurica was performed. The results indicated that the medicines contained many FFAs. Linear correlation coefficients for the FFA derivatives were >0.9991. Relative standard deviations (RSDs, n = 6) for the fatty acid derivatives were <3%. Detection limits (at a signal-to-noise ratio of 3:1) were 3.1–38 fmol. When the fatty acid derivatives were determined in the two real samples results were satisfactory and the sensitivity and reproducibility of the method were good.     

Evaluation of gas chromatographic methods for the determination of <Emphasis Type="Italic">trans</Emphasis> fat

Consumption of trans fat has been associated with increased risk of coronary heart disease. For nutrition labeling purposes, the US Food and Drug Administration (FDA) defines trans fat as the sum of all the fatty acids with at least one nonconjugated double bond in the trans configuration. The FDA regulation states that label declarations of trans fat are not required for products that contain less than 0.5 g of trans fat per serving if no claims are made about fat, fatty acids or cholesterol. While attenuated total reflection Fourier-transformed infrared spectroscopy (ATR-FT-IR) provides reproducible measurements for samples containing more than 5% trans fat, methods based on gas chromatography (GC) are needed to measure lower trans fat levels. Trans fat quantitation by GC has recently been updated by considering more fatty acids, focusing more attention on fatty acids present in low amounts, and by using 100-m high-polarity capillary columns for optimal separation. The consistently high interlaboratory relative standard deviations (RSD, e.g., 21% at 1% trans fatty acids (TFA), 60% at 0.17% TFA), and intralaboratory RSD values (e.g., 10% at 1% TFA, 16% at 0.17% TFA) for trans fat at 1% or less of total fat reported in the collaborative study data for American Oil Chemists Society Official Method Ce 1h-05 suggest the need to carefully define the parameters associated with GC analysis of fatty acids.     

Production of lipase by a newly isolated <Emphasis Type="Italic">Bacillus coagulans</Emphasis> under solid-state fermentation using melon wastes

Summary An extracellular lipase was produced by Bacillus coagulans by solid-state fermentation. Solid waste from melon was used as the basic nutrient source and was supplemented with olive oil. The highest lipase production (78,069 U/g) was achieved after 24h of cultivation with 1% olive oil enrichment. Enzyme had an optimal activity at 37°C and pH 7.0, and sodium dodecyl sulfate increased lipase activity. NH ₄NO₃ increased enzyme production, whereas organic nitrogen had no effect. The effect of the type of carbon sources on lipolytic enzyme production was also studied. The best results were obtained with starch and maltose (148,932 and 141,629 U/g, respectively), whereas a rather low enzyme activity was found in cultures grown on glucose and galactose (approx 118,769 and 123,622 U/g, respectively). Enzyme was inhibited with Mn⁺² and Ni⁺² by 68 and 74%, respectively. By contrast, Ca⁺² enhanced enzyme production by 5%.     

Rapid quantification of juvenile hormones and their metabolites in insect haemolymph by liquid chromatography–mass spectrometry (LC-MS)

A simple, fast and sensitive method was developed for routine determination of juvenile hormone (JH), JH diols and JH acids in insect haemolymph, by liquid chromatography–mass spectrometry (LC-MS). Sample clean-up involves the precipitation of proteins by methanol/isooctane (1:1, v/v), centrifugation and partial evaporation of the organic solvents. Since JH is bound to a carrier protein in the haemolymph, a binding protein (BP) assay was performed to ensure JH is removed during precipitation. The JH compounds were separated on a C₁₈ column (ReproSil-Pur ODS-3) by gradient elution with water and methanol in less than 22 min and analysed by electrospray mass spectrometry. Due to the high abundance of Na⁺ in insect haemolymph, [M+Na]⁺ is primarily formed. The limit of detection and quantification was 6 and 20 pg for JHs, and 8 and 25 pg for JH diols, respectively. To demonstrate the applicability of the method to different insect orders, haemolymph samples from the Mediterranean field cricket (Gryllus bimaculatus), the fall armyworm (Spodoptera frugiperda), the pea aphid (Acyrthosiphon pisum) and an ant species (Myrmicaria eumenoides) were analysed.Funded by the Deutsche Forschungsgemeinschaft (DFG) Graduate College 678: Ecological Significance of Natural Compounds and other Signals in Insects—from Structure to Function.Parts of this paper were presented at the 21st Conference of European Comparative Endocrinologists, 26–30 August 2002, Bonn, Germany     

Antibacterial mechanism of houttuyfonate homologues against Bacillus subtilis

In this study the effects of houttuyfonate homologues (HOU-C_n) on the surface and shape, the lipid fluidity and the protein conformation, the fatty acid compositions and fatty acid synthase II (FAS II) of B. subtilis were studied to elucidate the antibacterial mechanism of HOU-C_n against Bacillus subtilis. The scanning electronic microscope (SEM) results showed that the glycocalyx on the surface of B. subtilis disappeared and the cell became smaller after being treated with HOU-C_n. During the co-incubation of HOU-C_n and B. subtilis, HOU-C_n was decomposed into alkyl acyl aldehyde and sodium sulfite rapidly. Then alkyl acyl aldehyde was congregated onto cell membrane and inserted into lipid bilayer, further increased the fluidity of membrane and changed the conformation of membrane protein by hydrophobic binding. Subsequently, HOU-C_n inhibited the FAS II activity, and decreased the synthesis of fatty acids, especially increased the percentage of saturated fatty acid. HOU-C_n showed a strong antibacterial activity against G⁺-bacteria by a multi-target antibacterial mechanism.