Parameters affecting productivity in the lipase-catalysed synthesis of sucrose palmitate

The industrial application of lipases for the synthesis of sucrose esters is usually limited by its low productivity, as we need a medium where a polar reagent (the sugar) and a non-polar fatty acid donor are soluble and able to react in the presence of the biocatalyst. In this work, we have studied the problems encountered when trying to increase the volumetric productivity of sucrose esters. The synthesis of sucrose palmitate was performed in 2-methyl-2-butanol:dimethylsulfoxide mixtures by transesterification of different palmitic acid donors with sucrose, catalysed by the immobilized lipase from Candida antarctica B (Novozym 435). A protocol for substrate preparation different from that previously reported was found to improve the reaction rate. Several parameters, such as sucrose and acyl donor loadings, the percentage of DMSO in the mixture and the nature of acyl donor, were investigated. Under the best experimental conditions (15% DMSO, 0.1 mol l^?1 sucrose, 0.3 mol l^?1 vinyl palmitate), a maximum of 45 g l^?1 sucrose palmitate was obtained in 120 h. Using methyl or ethyl palmitate, the highest productivity was 7.3 g l^?1 in 120 h using 20% DMSO with 0.2 mol l^?1 sucrose and 0.6 mol l^?1 acyl donor. The formation of free fatty acid, and the effect of the percentage of DMSO on the monoester/diester selectivity were also studied. To our knowledge, this is the first report on enzymatic synthesis of sucrose esters of long fatty acids using alkyl esters as acyl donors.     

Lipid components of aristolochia longa

The hexane extract (non-volatiles) of Aristolochia longa yielded fatty acids, methyl, ethyl, isobutyl and phytyl esters, and polyprenols.     

酵母细胞表面展示技术及其在非水相酶催化合成中的应用

酵母展示技术是将外源蛋白与酵母细胞壁蛋白融合,并将外源蛋白表达在酵母细胞表面。酵母展示技术已广泛应用于各种功能蛋白的表达及筛选。以下重点介绍酵母展示技术在脂肪酶展示体系构建及其在脂肪酸甲酯、短链芳香酯及糖酯生物合成中的应用。     

Separation and analysis of steryl and wax esters from Dicranum elongatum

Abstract Complete separation of the steryl and wax esters in the subarctic moss Dicranum elongatum was achieved on MgO thin-layer plates without any notable alteration of the acyl and alkyl moieties of the esters. Gas chromatography-mass spectrometry of the hydrolyzed fraction showed that the sterols (campesterol, stigmasterol, sitosterol, cycloartenol, 24-methylene cycloartanol and an unidentified sterol) were primarily esterified with unsaturated fatty acids 18:2 ω 6, 18:3 ω 3 and 20:4 ω 6. In contrast, the wax alcohols (l-octadecanol, phytol and geranylgeraniol) were mainly esterified with saturated fatty acids with 16:0, 18:0 and 20:0 as major components. No great differences were found in the fatty acid pattern of the steryl esters between different portions of the shoot. Slight differences, however, were found in the proportions of ω 3 and ω 6 fatty acids. In the wax esters a clear decrease was found in the proportions of 18:0 and 20:0 acids with increased shoot age accompanied by a slight increase in the proportions of 14:0, 20:4 ω 6 and phytenic acid.     

Synthesis of deuterated methyl 6,9,12-octadecatrienoate geometric isomers

Methyl cis-6,cis-9,cis-12-octadecatrienoate-15,15,16,16-d₄ and the corresponding cis,cis,trans isomer were obtained by coupling hexyl-d₄-triphenylphosphonium bromide and methyl 12-oxo-cis-6,cis-9-dodecadienoate by the Wittig reaction. The deuterated phosphonium salt was prepared from 3-hexynol by catalytic deuteration of the corresponding tetrahydropyranyl ether and intermediate formation of the bromide. The dienoic aldehyde ester was obtained through the intermediate dioxanyl and dimethoxy derivatives from the Wittig coupling of methyl 9-oxo-cis-6-nonenoate with [2-(1,3-dioxan-2-yl)ethyl]-triphenylphosphonium bromide. The monoenoic aldehyde ester was prepared in a similar manner by the Wittig reaction between methyl 6-oxohexanoate and the dioxanylphosphonium salt. The saturated aldehyde ester was obtained, through several steps, from the ozonolysis of cyclohexene. Geometric isomers formed during each of the Wittig reactions were separated by silver resin chromatography. ¹³C Nuclear magnetic resonance chemical shifts for the compounds prepared are presented.     

Determination of retinol and retinyl esters in human plasma by high-performance liquid chromatography with automated column switching and ultraviolet detection

A HPLC method with automated column switching and UV detection is described for the simultaneous determination of retinol and major retinyl esters (retinyl palmitate, retinyl stearate, retinyl oleate and retinyl linoleate) in human plasma. Plasma (0.2 ml) was deproteinized by adding ethanol (1.5 ml) containing the internal standard retinyl propionate. Following centrifugation the supernatant was directly injected onto the pre-column packed with LiChrospher 100 RP-18 using 1.2% ammonium acetate–acetic acid–ethanol (80:1:20, v/v) as mobile phase. The elution strength of the ethanol containing sample solution was reduced by on-line supply of 1% ammonium acetate–acetic acid–ethanol (100:2:4, v/v). The retained retinol and retinyl esters were then transferred to the analytical column (Superspher 100 RP-18, endcapped) in the backflush mode and chromatographed under isocratic conditions using acetonitrile–methanol–ethanol–2-propanol (1:1:1:1, v/v) as mobile phase. Compounds of interest were detected at 325 nm. The method was linear in the range 2.5–2000 ng/ml with a limit of quantification for retinol and retinyl esters of 2.5 ng/ml. Mean recoveries from plasma were 93.4–96.5% for retinol (range 100–1000 ng/ml) and 92.7–96.0% for retinyl palmitate (range 5–1000 ng/ml). Inter-assay precision was ≤5.1% and ≤6.3% for retinol and retinyl palmitate, respectively. The method was successfully applied to more than 2000 human plasma samples from clinical studies. Endogenous levels of retinol and retinyl esters determined in female volunteers were in good accordance with published data.     

Optimization of alkyl ester production from grease using a phyllosilicate sol-gel immobilized lipase**,***

Simple alkyl ester derivatives of restaurant grease were prepared using a lipase from Pseudomonoas cepacia immobilized within a phyllosilicate sol-gel matrix as biocatalyst. Alcoholysis reactions of grease were carried out in solvent-free media using a one-step addition of alcohol to the reaction mixture. The immobilized lipase was active from 40 to 70 °C. Ester yields (60–97%) were highest when using a ratio of reactants of 2 mmol grease to 8 mmol alcohol and the biocatalyst was 10% (w/w) of grease in the presence of molecular sieves.     

Odoriferous compounds from the flowers of the conifers Picea abies,pinus sylvestris and Larix sibirica

A GC/MS analysis of the volatile constituents from the flowers of Norway Spruce, Picea abies, has been carried out. The volatile constituents of the female flowers were distinctly different from those of the male flowers and the twigs. Characteristic constituents are methyl and ethyl benzoate, methyl and ethyl salicylate, methyl and ethyl butanoate, borneol and bornyl acetate. In the scent from the male flowers we could only detect the same monoterpenes as in the twigs. In Larix sibirica methyl benzoate, methyl salicylate, borneol and bornyl acetate were detected in the female flowers and, in the female flowers of Pinus sylvestris, methyl salicylate was found.     

Selective retention of organic phosphate esters and phosphonates on aluminium oxide

Compounds containing the –PO₃H₂ function, such as monoesters of phosphoric acid and phosphonic acids, specifically bind to aluminium oxide in aqueous solution under experimental conditions where non-phosphorylated compounds are completely desorbed. The bound organic phosphate can be specifically displaced by aqueous solution of inorganic phosphates thus allowing their separation or detection by a technique similar to that of affinity chromatography. The consequences of this finding for phosphate compound biochemistry are discussed.     

Sterols and steryl esters in some Brassica and Sinapis seeds

The qualitative and quantitative composition of sterols in the free form and esterified to fatty acids was studied in seed oils from Brassica napus, B. campestris, B..iuncea, B. nigra, Sinapis alba and S. aruefisis (Brassica kaber). Sitosterol, followed by campesterol, predominated in both the free and the esterified sterols. The free sterols were richer in brassicasterol (ca 10–20%) than the steryl esters (3–10%). Small amounts of δ⁵-avenasterol and δ⁷-stigmastenol were also found in the Brassica oils, often more in the esterified than in the free form. The quantity of sterols was studied only in Brassica campestris, which had ca 0.3 % in the free as well as in the esterified form. In Sinapis alba, ca 10% of the sterols in the free form and 20 % in the esterified sterols were δ⁵-avenasterol. This compared to only a few per cent in both the free and esterified sterols in the Brassica oils. Similarly, ca 2 % of cholesterol was found among the sterols of Sinapis alba but only traces in the Brassica oils. The similarity of sterol compositions among the cultivated brassicas and wild mustard (Sinapis arvensis), and the specific characteristics of the sterols in white mustard (Sinapis alba) adds further weight to the suggestion that wild mustard should be treated as Brassica kaber and strengthens the generic separation of Sinapis alba.     

High-performance liquid chromatography of coenzyme A esters formed by transesterification of short-chain acylcarnitines: Diagnosis of acidemias by urinary analysis

A protocol for the identification and estimation of short-chain esters of carnitine is described; it is useful for the diagnosis of acidemias. By this method, carnitine esters in urine are converted to coenzyme A esters enzymatically with carnitine acetyltransferase (CAT): short-chain acylcarnitine + CoA cat in equilibrium short-chain acyl-CoA + carnitine. The coenzyme A esters are separated by high-performance liquid chromatography using a radial compression system with a C8 Radial-Pak cartridge and a mobile phase containing 0.025 M tetraethylammonium phosphate in a linear gradient of 1 to 50% methanol. Coenzyme A esters are quantitated by integrator determination of the area under the 254-nm absorption peaks. Enzymatic conversion approaches 100% for acetyl and propionyl esters except in the presence of high levels of free carnitine, which lowers the proportion of ester as acyl-CoA at equilibrium. However, since acidemia patients produce urine low in free carnitine, this problem is minimized. The method is rapid and simple and identifies propionic, methylmalonic, and isovaleric acidemias.     

Location and biosynthesis of monoterpenyl fatty acyl esters in rose petals

The upper epidermal layer of cells and the epicuticular wax surface of Lady Seton rose petals are sites of biosynthesis and accumulation, respectively, of a family of terpenyl fatty acyl esters. These esters are based mainly on the acyclic monoterpene alcohol geraniol coupled primarily to fatty acids of chain lengths 16-20 and in mass terms represent from 14% to 64% of the total monoterpenes present in the petals. The lipophilic nature of these non-volatile esters of the monoterpene alcohols contrasts with that of the lipophilic volatile parent alcohols themselves and with the hydrophilic, non-volatile, glucoside derivative of the other principal petal fragrant compounds, the phenylpropanoids, beta-phenyl ethanol and benzyl alcohol. These latter compounds are also synthesised and are resident in the petal. Biosynthetic studies confirmed that the petal upper epidermal cell layer has the capacity to incorporate mevalonic acid into the monoterpene component of the fatty acyl ester. The biosynthesis of the monoterpene component of the fatty acyl ester occurs via the mevalonic acid pathway in Lady Seton as well as in the hybrid tea rose Fragrant Cloud. In the latter flower the biosynthesis of geraniol was biosynthetically trans as was the formation of nerol and citronellol. Both geraniol and nerol were shown to be precursors of citronellol via an NADPH dependent reductase reaction. Oleic acid is assimilated into the acyl moiety of the terpenyl ester in Lady Seton isolated petal discs. It is probable that the lipophilic non-volatile terpenyl fatty acyl esters represent a stable storage form of the corresponding alcohols from their residency within the epicuticular wax layer. These acyl esters may realise, on hydrolysis, additional aroma notes from the living flower and potentially commercially significant quantities of the fragrant terpenols during oil of rose essence production.     

Selective synthesis of panthenyl esters by a kinetically controlled enzymatic process

Abstract

Panthenyl esters (panthenyl monoacetate and panthenyl diacetate) were synthesized in high yields (≈100%) by a kinetic reaction control using a commercial immobilized Candida Antarctica lipase B (Novozyme 435) in acetonitrile. The enzyme showed excellent synthetic activity, regioselectivity, and operational stability under the conditions used.     

Purification and Characterization of an Esterase from Micrococcus sp. YGJ1 Hydrolyzing Phthalate Esters

An esterase hydrolyzing phthalate esters has been purified from Micrococcus sp. YGJ1. The enzyme, a monomeric protein (M_r=56 kDa) with a pI of 4.0, hydrolyzes various aliphatic and aromatic carboxylesters. The medium chain (C₃-C₄) esters are the most preferred substrates. The enzyme is inhibited by HgCl₂ and p-chloromercuribenzoate but not by phenylmethyl-sulfonyl fluoride.     

Rapid quantitation of free fatty acids in human plasma by high-performance liquid chromatography

We report a rapid and sensitive method for separation and quantitation of free fatty acids (FFAs) in human plasma using high-performance liquid chromatography (HPLC). Two established techniques of lipid extraction were investigated and modified to achieve maximal FFA recovery in a reasonably short time period. A modified Dole extraction method exhibited greater recovery (90%) and short processing times (30 min) compared to the method of Miles et al. Reversed-phase HPLC using UV detection was used for plasma FFA separation and quantitation. Two phenacyl ester derivatives, phenacyl bromide and p-bromophenacyl bromide, were investigated in order to achieve optimal separation of individual plasma FFAs (saturated and unsaturated) with desirable detection limits. Different chromatographic parameters including column temperature, column type and elution profiles (isocratic and gradient) were tested to achieve optimal separation and recovery of fatty acids. Phenacyl bromide esters of plasma fatty acids were best resolved using an octadecylsilyl column with endcapped silanol groups. An isocratic elution method using acetonitrile–water (83:17) at 2 ml/min with UV detection at 242 nm and a column temperature of 45°C was found to optimally resolve the six major free fatty acids present in human plasma (myristic [14:0], palmitic [16:0], palmitoleic [16:1], stearic [18:0], oleic [18:1] and linoleic [18:2]), with a run time of less than 35 min and detection limits in the nmol range. The entire process including plasma extraction, pre-column derivatization, and HPLC quantitation can be completed in 90 min with plasma samples as small as 50 μl. Over a wide physiological range, plasma FFA concentrations determined using our HPLC method agree closely with measurements using established TLC–GC methods (r²≥0.95). In addition, by measuring [¹⁴C] or [³H] radioactivity in eluent fractions following HPLC separation of plasma FFA, this method can also quantitate rates of FFA turnover in vivo in human metabolic studies employing isotopic tracers of one or more fatty acids.     

Purification of fatty acid methyl esters by high-performance liquid chromatography

The determination by gas chromatography (GC) of fatty acid methyl esters (FAMEs) prepared from complex biological samples is subject to interference from cholesterol. During sample injection on the GC system of FAMEs prepared from tissues that contain cholesterol, we observed a major contaminant that co-eluted with docosahexaenoic acid (DHA, 22:6n-3). To address this problem, FAMEs were purified on an amino-phase high-performance liquid chromatography (HPLC) column using a hexane–isopropanol gradient. The HPLC retention times for both the FAME fraction and cholesterol were stable and reproducible when the amino column was used for sample purification. The purified extracts were analyzed by GC without artifacts or impurity peaks after 50 analytical runs. The method described here will be useful for measurement of 22:6n-3 and other fatty acids important for studies of nutrition or pathology.     

Design and synthesis of novel antimicrobials with activity against Gram-positive bacteria and mycobacterial species,including M. tuberculosis

The alarming increase in bacterial resistance over the last decade along with a dramatic decrease in new treatments for infections has led to problems in the healthcare industry. Tuberculosis (TB) is caused mainly by Mycobacterium tuberculosis which is responsible for 1.4 million deaths per year. A world-wide threat with HIV co-infected with multi and extensively drug-resistant strains of TB has emerged. In this regard, herein, novel acrylic acid ethyl ester derivatives were synthesized in simple, efficient routes and evaluated as potential agents against several Mycobacterium species. These were synthesized via a stereospecific process for structure activity relationship (SAR) studies. Minimum inhibitory concentration (MIC) assays indicated that esters 12, 13, and 20 exhibited greater in vitro activity against Mycobacterium smegmatis than rifampin, one of the current, first-line anti-mycobacterial chemotherapeutic agents. Based on these studies the acrylic ester 20 has been developed as a potential lead compound which was found to have an MIC value of 0.4 μg/mL against Mycobacterium tuberculosis. The SAR and biological activity of this series is presented; a Michael-acceptor mechanism appears to be important for potent activity of this series of analogs.     

Facile one-pot mono-dealkylation of H-phosphonate esters in high yield

The readily available dialkyl H-phosphonates (RO)₂P(O)H, where R = Me, Et, ⁱPr, ⁿBu and Bn can be mono-dealkylated by heating at reflux in excess tert-butylamine. Where R = Me or Bn then crystals of [(CH₃)₃CNH₃]⁺ [ROP(O)HO]⁻ form in the reaction vessel on overnight standing.     

Immunochemical recognition of phosphate ester derivatives of phenol rings is dependent upon the electronic charge of the ester group

The recognition of phosphate and sulphate esters of tyrosine residues has been studied employing antisera with specificity for tyrosine phosphate, and the enzymes aryl sulphatase, and acid and alkaline phosphatases. The ability of tyrosine phosphate, and of phosphate esters of phenol, to inhibit the antiserum was pH dependent. The capacity to effect inhibition appeared to correlate with alterations in the ionisation of the inhibitor. Moreover, the antisera with reactivity for tyrosine phosphate had no reactivity with tyrosine sulphate or sulphate esters of phenol at any pH value studied. The enzymes alkaline phosphatase, acid phosphatase, and aryl sulphatase were also studied. The phosphatases were found not to hydrolyse sulphate ester containing substrate analogues at any pH value in the range 5.0–9.0. In contrast, aryl sulphatase appeared to hydrolyse phosphate esters at pH 5.0 and 7.0, but not at pH 9.0.Abbreviations ABP Azobenzyl phosphonate - KLH-ABP Keyhole limpet haemocyanin derivatised with azobenzyl phosphonate groups - OVA-ABP Ovalbumin derivatised with azobenzylphosphonate groups