Lipase-catalysed synthesis of glucose fatty acid esters in tert-butanol

Synthesis of 6-O-acylate--d-glycopyranose from underivatised substrates in anhydrous tert-butanol was achieved using immobilised lipases from Candida antarctica and Mucor miehei. Except for acetic acid, the initial reaction rates with the C. antarctica lipase were independent of acyl donor chain lengths and in a range of 3.9±0.4 mol glucose converted min–1 g enzyme preparation. The catalytic activity of the M. miehei lipase increased with increasing acyl donor chain length with a maximum for stearic acid of 0.45 mol min–1 g. Using maltose as substrate, the catalytic activity decreased by a factor of 48 and 20 with the lipase from C. antarctica and M. miehei, respectively, while with maltotriose no reaction was observed.     

Continuous refolding of lysozyme with fed-batch addition of denatured protein solution

A continuous refolding method with addition of denatured protein solution in a fed-batch manner through a ceramic membrane tube was developed. Denatured and fully reduced lysozyme was continuously refolded with high refolding efficiencies. In this method, a denatured lysozyme solution was gradually added from the outer surface of the membrane tube into a refolding buffer flowing continuously inside the tube under controlled mixing conditions. The refolding efficiencies of lysozyme in this continuous refolding were higher than those in a batch dilution method. This method has applicability to large-scale downstream processes and can attain a high efficiency and protein concentration in refolding. Refolded proteins can be supplied continuously following purification steps.     

Enzymatic synthesis of terpenyl esters by transesterification with fatty acid vinyl esters as acyl donors by Trichosporon fermentans lipase

Enzymatic synthesis of terpenyl esters by esterification or transesterification with fatty acid vinyl esters as acyl donors by celite-adsorbed lipase of Trichosporon fermentans was investigated. In direct esterification of geraniol, the lipase showed high reactivity toward fatty acids with carbon chains longer than C-8, but little reactivity toward fatty acids with shorter chains. With fatty acid vinyl esters as acyl donors, the lipase catalysed the synthesis of geranyl and citronellyl esters with carbon chains shorter than C-6 in with yields of >90% molar conversion. Time course, effects of added water, temperature and substrate concentration were studied for the synthesis of geranyl acetate. Molar conversion yield reached 97.5% after 5 h incubation at 30–40°C with the addition of 3% water. In this reaction, no inhibition by substrates such as geraniol and vinyl acetate was observed.     

Determination of regioisomeric distribution in carbohydrate fatty acid monoesters by LC-ESI-MS

A new LC-ESI-MS method for characterizing the regioisomeric distribution in carbohydrate monoesters with long-chain fatty acids is described. Sucrose monolaurate mixtures were used for development of the method. The surfactant nature and high polarity of these compounds make them appropriate analytes for being detected by electrospray-ionization mass spectrometry (ESI-MS). Despite the structural similarity of regioisomers, an excellent resolution of all regioisomers present in the different samples studied (sucrose monodecanoates, sucrose monolaurates, sucrose monopalmitates and melezitose monolaurates) is achieved. Reversed-phase liquid chromatography with isocratic acetonitrile-water mixtures was used for a proper separation of the analytes. Finally, the superiority of this chromatographic method for determining the regioselectivity in enzymatic carbohydrate acylation reactions, with respect to the typical methodology based on routine 13C NMR spectroscopy, is also discussed.     

Refolding of denatured/reduced lysozyme at high concentrations by artificial molecular chaperone‐ion exchange chromatography

Development of high efficiency and low cost protein refolding methods is a highlighted research focus in biotechnology. Artificial molecular chaperone (AMC) and protein folding liquid chromatography (PFLC) are two attractive refolding methods developed in recent years. In the present work, AMC and one branch of PFLC, ion exchange chromatography (IEC), are integrated to form a new refolding method, artificial molecular chaperone‐ion exchange chromatography (AMC‐IEC). This new method is applied to the refolding of a widely used model protein, urea‐denatured/dithiothreitol‐reduced lysozyme. Many factors influencing the refolding of lysozyme, such as urea concentration, β‐cyclodextrin concentration, molar ratio of detergent to protein, mobile phase flow rate, and type of detergent, were investigated, respectively, to optimize the conditions for lysozyme refolding by AMC‐IEC. Compared with normal IEC refolding method, the activity recoveries of lysozyme obtained by AMC‐IEC were much higher in the investigated range of initial protein concentrations. Moreover, the activity recoveries obtained by using this newly developed refolding method were still quite high for denatured/reduced lysozyme at high initial concentrations. When the initial protein concentration was 200 mg mL^?1, the activity recovery was over 60%. In addition, the lifetime of the chromatographic column during AMC‐IEC was much longer than that during protein refolding by normal IEC. Therefore, AMC‐IEC is a high efficient and low cost protein refolding method. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Synergistic cosolubilization of omega-3 fatty acid esters and CoQ10 in dilutable microemulsions

Water-dilutable microemulsions were prepared and loaded with two types of omega-3 fatty acid esters (omega-3 ethyl esters, OEE; and omega-3 triacylglycerides, OTG), each separately and together with ubiquinone (CoQ₁₀). The microemulsions showed high and synergistic loading capabilities. The linear fatty acid ester (OEE) solubilization capacity was greater than that of the bulky and robust OTG.The location of the guest molecules within the microemulsions at any dilution point were determined by electrical conductivity, viscosity, DSC, SAXS, cryo-TEM, SD-NMR, and DLS.We found that OEE molecules pack well within the surfactant tails to form reverse micelles that gradually, upon water dilution, invert into bicontinuous phase and finally into O/W droplets. The CoQ₁₀ increases the stabilization and solubilization of the omega-3 fatty acid esters because it functions as a kosmotropic agent in the micellar system. The hydrophobic and bulky OTG molecule strongly interferes with the tail packing and spaces them significantly – mainly in the low and medium range water dilutions. When added to the micellar system, CoQ₁₀ forms some reverse hexagonal mesophases. The inversion into direct micelles is more difficult in comparison to the OEE system and requires additional water dilution. The OTG with or without CoQ₁₀ destabilizes the structures and decreases the solubilization capacity since it acts as a chaotropic agent to the micellar system and as a kosmotropic agent to hexagonal packing. These results explain the differences in the behavior of these molecules with vehicles that solubilize them in aqueous phases.Temperature disorders the bicontinuous structures and reduces the supersaturation of the system containing OEE with CoQ₁₀; as a result CoQ₁₀ crystallization is retarded.     

Growth inhibitory properties of lactose fatty acid esters

Sugar esters are biodegradable, nonionic surfactants which have microbial inhibitory properties. The influence of the fatty acid chain length on the microbial inhibitory properties of lactose esters was investigated in this study. Specifically, lactose monooctanoate (LMO), lactose monodecanoate (LMD), lactose monolaurate (LML) and lactose monomyristate (LMM) were synthesized and dissolved in both dimethyl sulfoxide (DMSO) and ethanol. Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) were determined in growth media. LML was the most effective ester, exhibiting MIC values of <0.05 to <5 mg/ml for each Gram-positive bacteria tested (Bacillus cereus, Mycobacterium KMS, Streptococcus suis, Listeria monocytogenes, Enterococcus faecalis, and Streptococcus mutans) and MBC values of <3 to <5 mg/ml for B. cereus, M. KMS, S. suis, and L. monocytogenes. LMD showed MIC and MBC values of <1 to <5 mg/ml for B. cereus, M. KMS, S. suis, L. monocytogenes, and E. faecalis, with greater inhibition when dissolved in ethanol. LMM showed MIC and MBC values of <1 to <5 mg/ml for B. cereus, M. KMS, and S. suis. LMO was the least effective showing a MBC value of <5 mg/ml for only B. cereus, though MIC values for S. suis and L. monocytogenes were observed when dissolved in DMSO. B. cereus and S. suis were the most susceptible to the lactose esters tested, while S. mutans and E. faecalis were the most resilient and no esters were effective on Escherichia coli O157:H7. This research showed that lactose esters esterified with decanoic and lauric acids exhibited greater microbial inhibitory properties than lactose esters of octanoate and myristate against Gram-positive bacteria.     

Microwave-assisted synthesis of deuterium labeled estrogen fatty acid esters

Deuterated analogs of estrogen fatty acid esters are needed as internal standards for isotope dilution GC/MS analyses. We have developed a rapid and efficient synthesis for 2,4,16,16-D4-estrone palmitate, stearate, oleate, linoleate, and linolenate and the corresponding 2,4,16,16,17alpha-D5-estradiol fatty acid 17-mono and 3,17-diesters using analogous fatty acid chlorides or fatty acid anhydrides and 4-(dimethylamino)pyridine under microwave irradiation. Chemoselective hydrolysis of fatty acid diesters was carried out by KOH in t-BuOH.     

Porcine pancreatic lipase hydrophobically adsorbed on octyl-silica: A robust biocatalyst for syntheses of xylose fatty acid esters

This work describes the immobilization of porcine pancreatic lipase (PPL), obtained from crude extract, on silica coated with octyl groups (OS) by interfacial adsorption, a simple and low-cost immobilization protocol. The biocatalyst PPL-OS was employed to the enzymatic preparation of fatty acid esters of d-xylose, a product used especially in the field of cosmetics and pharmacy, especially dermatology, improving the functionality of epidermal cells. The yields of the immobilization in terms of enzymatic activity and protein concentration (98% and 75%, respectively) suggested that PPL present in the crude extract was selectively immobilized on the octyl-silica support, which allowed the hyperactivation of the biocatalyst (recovered activity, 144%), a phenomenon that may be attributed to the interfacial activation of the enzyme on hydrophobic surfaces. The biocatalyst PPL-OS showed to be very robust in organic medium and at high temperature, which is an extremely important characteristic to produce sugar fatty acid esters from the industrial point of view. The syntheses of xylose fatty esters (oleate, caprylate and butyrate) yielded conversions around 70% after short reaction period (2?h) at 60?°C in tert-butyl alcohol. The biocatalyst, even after incubation at 60?°C for 24?h, could be reused in four esterification cycles of 2-h reaction at 60?°C, maintaining 100% of its catalytic activity.     

Synthesis and properties of fatty acid starch esters

Being completely bio-based, fatty acid starch esters (FASEs) are attractive materials that represent an alternative to crude oil-based plastics. In this study, two synthesis methods were compared in terms of their efficiency, toxicity and, especially, product solubility with starch laurate (C₁₂) as model compound. Laurates (DS > 2) were obtained through transesterification of fatty acid vinylesters in DMSO or reaction with fatty acid chlorides in pyridine. The latter lead to higher DS-values in a shorter reaction time. But due to the much better solubility of the products compared to lauroyl chloride esterified ones, vinylester-transesterification was preferred to optimize reaction parameters, where reaction time could be shortened to 2 h. FASEs C₆–C₁₈ were also successfully prepared via transesterification. To determine the DS of the resulting starch laurates, the efficient ATR-IR method was compared with common methods (elementary analysis, ¹H NMR). Molar masses (M_w) of the highly soluble starch laurates were analyzed using SEC-MALLS (THF). High recovery rates (>80%) attest to the outstanding solubility of products obtained through transesterification, caused by a slight disintegration during synthesis. Particle size distributions (DLS) demonstrated stable dissolutions in CHCl₃ of vinyl laurate esterified – contrary to lauroyl chloride esterified starch. For all highly soluble FASEs (C₆–C₁₈), formation of concentrated solutions (10 wt%) is feasible.     

Effects of acetyl-L-carnitine on the formation of fatty acid ethyl esters in brain and peripheral organs after short-term ethanol administration in rat

Increasing evidence suggests that Fatty acid ethyl esters (FAEE) play a central role in ethanol induced organ damage. In the current study we measured FAEE formation in rats after short-term oral administration of ethanol, in the presence and absence of pre-treatment with acetyl-L-carnitine. Ethanol treatment caused a significant increase in the levels of FAEE, particularly in the brain and heart, but also in the kidney and liver. Increases in FAEE were associated with a significant increase in FAEE synthase activity, GSH transferase activity, and lipid hydroperoxide levels. Pre-treatment with acetyl-L-carnitine resulted in a significant reduction of FAEE accumulation, decrease in FAEE synthase and GSH transferase activities, and lipid hydroperoxide levels. Administration of acetyl-L-carnitine greatly reduced the metabolic abnormalities due to non-oxidative ethanol metabolism, through an increment in lipid metabolism/turnover and by the modulation of the activities of enzymes associated with FAEE synthesis. These results suggest a potentially important pharmacological role for acetyl-L-carnitine in the prevention of alcohol-induced cellular damage.     

Fatty acid analysis of triacylglycerols: Preparation of fatty acid methyl esters for gas chromatography

A method to prepare fatty acid methyl esters was developed for fatty acid analysis of triacylglycerols by gas chromatography (GC). Triacylglycerols were mixed with methanolic CH₃ONa in hexane containing a mid-polar solvent for 10 s at room temperature. Under these conditions, trioleoylglycerol was converted to methyl oleate with an average yield of 99.3%. This procedure gave reliable and reproducible data on fatty acid compositions determined by GC.     

Lipoxygenase inhibition by novel fatty acid ester from Annona squamosa seeds

Studies on the seeds of Annona squamosa yielded a novel lipoxygenase inhibitor fatty acid ester, (+) - annonlipoxy (1). Compound 1 was screened for its enzyme inhibitory activity against lipoxygenase (E.C.1.14.18.1), exhibiting activity with IC₅₀ 69.05 ± 5.06 μm. Baicalein (IC₅₀ 22.6 ± 0.5 μm) was used as a positive control. Crude extracts of Annona squamosa fruit pulp and seeds were screened for its enzyme inhibitory activity against lipoxygenase and acetylcholinesterase. The crude ethanolic extract of fruit pulp and seeds of Annona squamosa also exhibited lipoxygenase activity with 22.2 and 26.7% inhibition, while the pet.ether extract of seeds of A. squamosa exhibited 52.7% inhibition at a concentration of 40 μg/200 ml. The crude ethanolic extract of seeds of Annona squamosa was also bioassayed for acetylcholinesterase inhibition and it was found inactive.     

Effects of gibberellic acid on sucrose accumulation and sucrose biosynthesizing enzymes activity during banana ripening

During banana ripening there is a massive conversion into sugars, mainly sucrose, which can account for more than 10% of the fresh weight of the fruit. An ethylene burst is the trigger of the banana ripening process but there is evidence that other compounds can act as modulators of some biochemical pathways. As previously demonstrated, gibberellic acid (GA₃) can impair the onset of starch degradation and affect some degradative enzymes, but effects on the sucrose biosynthetic apparatus have not yet been elucidated. Here, the activity and amount of sucrose synthase (SuSy; E.C. 2.4.1.13) and sucrose–phosphate synthase (SPS; E.C. 2.4.1.14), respiration rates, ethylene production, and carbohydrate levels, were evaluated in GA₃-infiltrated and non-infiltrated banana slices. The exogenous supply of gibberellin did not alter the respiration or the ethylene profile but delayed sucrose accumulation by at least 2 days. While SuSy activity was similar in control and treated slices, SPS increase and sucrose accumulation was related in treated slices. Western blotting with specific antiserum showed no apparent effects of GA₃ on the amount of SuSy protein, but impaired the increase in SPS protein during ripening. The overall results indicate that although GA₃ did not block carbohydrate mobilisation in a irreversibly way, it clearly affected the triggering of starch breakdown and sucrose synthesis. Also, the delayed sucrose accumulation in GA₃-infiltrated slices could be explained by the disturbance of SPS activity. In conclusion, gibberellins can play an important role during banana ripening and our results also reinforce the idea of multiple regulatory components in the ripening pathway, as evidenced by the GA₃ effects.     

Structural characterization of saturated branched chain fatty acid methyl esters by collisional dissociation of molecular ions generated by electron ionization

Saturated branched chain fatty acids (BCFA) are present as complex mixtures in numerous biological samples. The traditional method for structure elucidation, electron ionization (EI) mass spectrometry, sometimes does not unambiguously enable assignment of branching in isomeric BCFA. Zirrolli and Murphy (Zirrolli , J. A. , and R. A. Murphy. 1993. Low-energy tandem mass spectrometry of the molecular ion derived from fatty acid methyl esters: a novel method for analysis of branched-chain fatty acids. J. Am. Soc. Mass Spectrom. 4: 223–229.) showed that the molecular ions of four BCFA methyl ester (BCFAME) yield highly characteristic fragments upon collisional dissociation using a triple quadrupole instrument. Here, we confirm and extend these results by analysis using a tabletop 3-D ion trap for activated molecular ion EI-MS/MS to 30 BCFAME. iso-BCFAME produces a prominent ion (30-100% of base peak) for [M-43] (M-C₃H₇), corresponding to the terminal isopropyl moiety in the original iso-BCFAME. Anteiso-FAME yield prominent ions (20-100% of base peak) corresponding to losses on both side of the methyl branch, [M-29] and [M-57], and tend to produce more prominent m/z 115 peaks corresponding to a cyclization product around the ester. Dimethyl and tetramethyl FAME, with branches separated by at least one methylene group, yield fragment on both sides of the sites of methyl branches that are more than 6 C away from the carboxyl carbon. EI-MS/MS yields uniquely specific ions that enable highly confident structural identification and quantification of BCFAME.     

Estimation of short-chain fatty acid production from protein by human intestinal bacteria based on branched-chain fatty acid measurements

Abstract The importance of protein breakdown and amino acid fermentation in the overall economy of the large intestine has not been quantitated. We have therefore measured the production of branched chain-fatty acids (BCFA) both in vitro and in vivo in order to estimate the contribution of protein to fermentation.
In vitro batch-culture studies using human faecal inocula showed that short-chain fatty acids (SCFA) were the principal end products formed during the degradation of protein by human colonic bacteria. Approximately 30% of the protein broken down was converted to SCFA. Branched-chain fatty acids (BCFA) constituted 16% of the SCFA produced from bovine serum albumin and 21% of the SCFA generated when casein was the substrate. BCFA concentrations in gut contents taken from the human proximal and distal colons were on average, 4.6 and 6.3 mmol kg⁻¹ respectively, corresponding to 3.4% and 7.5% of the total SCFA. These results suggest that protein fermentation could potentially account for about 17% of the SCFA found in the caecum, and 38% of the SCFA produced in the sigmoid/rectum. Measurements of BCFA in portal and arterial blood taken from individuals undergoing emergency surgery indicated that net production of BCFA by the gut microflora was in the region of 11.1 mmol day⁻¹, which would require the breakdown of about 12 g of protein. These data highlight the role of protein in the colon and may explain why many colonic diseases affect mainly the distal bowel.     

Estimation of short-chain fatty acid production from protein by human intestinal bacteria based on branched-chain fatty acid measurements

Abstract The importance of protein breakdown and amino acid fermentation in the overall economy of the large intestine has not been quantitated. We have therefore measured the production of branched chain-fatty acids (BCFA) both in vitro and in vivo in order to estimate the contribution of protein to fermentation.
In vitro batch-culture studies using human faecal inocula showed that short-chain fatty acids (SCFA) were the principal end products formed during the degradation of protein by human colonic bacteria. Approximately 30% of the protein broken down was converted to SCFA. Branched-chain fatty acids (BCFA) constituted 16% of the SCFA produced from bovine serum albumin and 21% of the SCFA generated when casein was the substrate. BCFA concentrations in gut contents taken from the human proximal and distal colons were on average, 4.6 and 6.3 mmol kg⁻¹ respectively, corresponding to 3.4% and 7.5% of the total SCFA. These results suggest that protein fermentation could potentially account for about 17% of the SCFA found in the caecum, and 38% of the SCFA produced in the sigmoid/rectum. Measurements of BCFA in portal and arterial blood taken from individuals undergoing emergency surgery indicated that net production of BCFA by the gut microflora was in the region of 11.1 mmol day⁻¹, which would require the breakdown of about 12 g of protein. These data highlight the role of protein in the colon and may explain why many colonic diseases affect mainly the distal bowel.     

Biodegradation of diesel oil and production of fatty acid esters by a newly isolated Pseudomonas citronellolis KHA

The ability of a newly isolated Pseudomonas citronellolis KHA to degrade diesel oil and to synthesize fatty acid esters has been screened in aerobic batch cultures. The microorganism was able to grow with diesel oil at initial concentrations up to 126 g/l, with optimal growth at 25 g/l. Strain KHA has produced compounds showing strong emulsifying properties (E₂₄ = 75% at the end of the exponential growth phase). The crude extract reduces the surface tension of water from 72 mN m⁻¹ down to 35 mN m⁻¹ with a corresponding minimal concentration value of 60 mg/l. GC and GC–MS analysis of crude product show that the major components are those of hexadecanoic acid propyl ester and octadecanoic acid propyl ester, which have potential for applications in cosmetics, pharmaceutical and foods industries. In addition, strain KHA represents a valuable source of compounds with surface-active properties and potential for the application in clean up of the sites contaminated with hydrocarbons.     

Alkylthiolation for the determination of double-bond position in unsaturated fatty acid esters

The methyl esters of some mono-unsaturated fatty acids have been methylthiolated by the iodine-catalysed addition of dimethyl disulfide across the double bond. The resulting derivatives are suitable for gas chromatography. The fragmentation of the derivatives on electron impact yields mass spectra which allow immediate recognition of the position of the original double bond.