Enzymatic synthesis of new aromatic and aliphatic esters of flavonoids using Candida antarctica lipase as biocatalyst

Enzymatic acylation of rutin and esculin with aromatic, aliphatic and aryl-aliphatic acids using Candida antarctica lipase in tert amyl alcohol as solvent was investigated under low water content. Whatever the acyl donor used, the conversion yields and initial rates for esculin were higher than for rutin. For a given flavonoid, the performance of these reactions depended on the acyl donor structures. For aliphatic acids, conversion yields and initial rates of both flavonoids were respectively in the ranges of 68–90% and of 9.5×10^?2–72×10^?2 mmol l^?1 h^?1. For aromatic acids, the reaction occurred only with the aryl subgroup (cinnamic, hydrocinnamic, 3,4-dihydroxyhydrocinnamic and 4-hydroxyphenyl acetic acids) and was drastically influenced by the presence of side chain and substitution patterns of the aromatic ring. Except for hydrocinnamic acid (75%, 23.4×10^?2 mmol l^?1 h^?1), with these acids the conversion yields and initial rates were lower and in the range of 10–45% and of 0.7×10^?2 to 12.1×10^?2 mmol l^?1 h^?1. Unsaturation of the side chain of the hydrocinnamic acid decreased the esculin conversion rate from 75 to 13% and initial rate from 23.4 to 1.76×10^?2 mmol l^?1 h^?1. The presence of hydroxyl or nitro-groups on the aromatic ring of the aryl aliphatic acid also reduced conversion yields and initial rates. Even without a spacer, the non-phenolic ring acid (quinic acid) was reactive and lead to conversion yields of about 20 and 23% respectively for rutin and esculin.     

Effect of acyl donor chain length and substitutions pattern on the enzymatic acylation of flavonoids

Rutin and esculin were enzymatically acylated with different aliphatic acids as acyl donors (fatty acids, dicarboxylic acids and ω-substituted fatty acids) by an immobilized lipase from Candida antarctica. The effect of the water content and the acyl donors pattern on the flavonoid initial acylation rate and conversion yield were investigated. The obtained results indicated that the water content of the medium has a strong effect on the performance of these reactions. The best conversion yields were reached when the water content was kept lower than 200 ppm. At low water content of the medium, these syntheses are influenced by carbon chain length and substitution pattern of the acyl donors. Higher conversion yields of esculin and rutin (>70%) were obtained with aliphatic acids having high carbon chain length (>12). Moreover, it has been found that the amine and thiol groups on ω-substituted fatty acid chain were unfavourable to these reactions. The ¹H NMR and ¹³C NMR analyses of some synthesized esters (esculin and rutin palmitate) show that only monoesters were produced and that the esterification takes place on the primary OH of glucose moiety of the esculin and on the secondary 4′′′-OH of the rhamnose residue of rutin.     

Acylation of natural flavonoids using lipase of candida antarctica as biocatalyst

Several flavonoids (quercetin, hesperidin, rutin and esculin) were acylated with fatty acids using an immobilised lipase from Candida antarctica in 2-methyl-2-butanol at 60 °C. It appears that esculin with primary OH on the sugar part is the most reactive substrate. With palmitic acid as acyl donor, the conversion yields were of about 80, 71 and 38%, respectively, for esculin, rutin and hesperidin. No reaction was observed with aglycon flavonoid (quercetin). For a given flavonoid (rutin), the conversion yield increased from 42 to 76% when the carbon number of the fatty acids rose from C6 to C12. For fatty acids with higher carbon-chain length, both conversion yield and initial rate dropped slightly. Furthermore, compared to the saturated fatty acid (C18: 0), the unsaturated one (C18: 1) exhibited a lower reactivity. For all molecules studied ¹H nuclear magnetic resonance (NMR) and ¹³C NMR analyses indicated that only flavonoid monoester was produced.     

Kinetics of Pyruvate Decarboxylase Deactivation by Benzaldehyde

Based on experimental data, a kinetic model for the deactivation of partially purified pyruvate decarboxylase (PDC) by benzaldehyde (0-200 mM) in MOPS buffer (2.5 M) has been developed. An initial lag period prior to deactivation was found to occur. With first order dependencies of PDC deactivation on exposure time and on benzaldehyde concentration, a reaction time deactivation constant of 2.64×10^-3 h^-1 and a benzaldehyde deactivation coefficient of 1.98×10^-4 mM^-1 h^-1 were determined for benzaldehyde concentrations up to 200 mM. The PDC deactivation kinetic equations established in this study are an essential component in an overall model being developed to describe the enzymatic biotransformation of benzaldehyde and pyruvate to produce the pharmaceutical intermediate (R)-phenylacetylcarbinol (R-PAC).     

Inhibition of antennal esterases of the egyptian armyworm Spodoptera littoralis by trifluoromethyl ketones

A series of aliphatic and aromatic trifluoromethyl ketones has been tested as inhibitors of the antennal esterases of the Egyptian armyworm Spodoptera littoralis, by evaluation of the extent of hydrolysis of [1-³H]-(Z,E)-9, 11-tetradecadienyl acetate (1), a tritiated analog of the major component of the sex pheromone. The most active compounds with a long chain aliphatic structure were 3-octylthio-1,1,1-trifluoropropan-2-one (2) (IC₅₀ 0.55 μM) and 1,1,1-trifluorotetradecan-2-one (4) (IC₅₀ 1.16 μM). The aromatic compounds were generally less potent inhbitors than the coressponding aromatic ones, although β-naphthyltrifuloromethyl ketone (10) exhibited a remarkable inhibitory activity (IC₅₀ 7.9 μM). Compounds 2, 4 and 10 exhibit a competitive inhibition with K_i values of 2.51×10⁻⁵ M, 2.98×10⁻⁵ M and 2.49×10⁻⁴ M, respectively. Some of the trifluoromethyl ketones tested were slow-binding inhibitors and compounds 2 and 10 are described as inhibitors of the antennal esterases of a moth for the first time.     

The potential role of microzooplankton in a northwestern Australian pelagic food web

The role of microzooplankton in waters adjacent to Australia's North West Cape (21°49'S 114°14'E) was studied during the austral summers 1997/1998 and 1998/1999. We estimated microzooplankton abundance and biomass at a shallow (∼20 m) shelf station and at a shelf break station (∼80 m). Microzooplankton were placed into six categories: four ciliate groups (strombidiids, strobilidiids, tintinnids, “other ciliates”), dinoflagellates, and sarcodines. Total microzooplankton abundances ranged between 0.14×10³ l^-1 and 3.4×10³ l^-1. The most abundant groups were the dinoflagellates (mean 459±73 standard error l^-1) and strombidiids (mean 334±42 standard error l^-1). Total microzooplankton biomass ranged between 0.03 and 1.70 µg C l^-1 (mean 0.33±0.05 standard error l^-1). Redundancy analysis indicated differences in microzooplankton community composition between stations and sampling years but no differences with sampling depth. The microzooplankton community showed considerable variability between adjacent sampling dates, reinforcing the conclusion of earlier studies that this area is a dynamic environment. Ciliate production on the shelf was estimated to be 1.05 µg C l^-1day^-1 (∼20 mg C m^-2day^-1) and 0.79 µg C l^-1day^-1(∼70 mg C m^-2day^-1) at the shelf break. Ciliate production near North West Cape was two- to six-fold higher than the rate of secondary production by juvenile copepods. Despite this, ciliate grazing appears to account for only ∼5% of primary production and ciliates do not appear to be a major conduit between primary producers and higher trophic levels in these waters.     

虎斑乌贼的胚胎耗氧率

为了探究虎斑乌贼胚胎不同发育时期的耗氧率变化和几种生态因子对胚胎发育过程耗氧率的影响,试验采用封闭静水装置,对不同发育时期(12期)的耗氧率进行测定,并研究不同盐度(21、24、27、30、33)、温度(18、21、24、27、30 ℃)和pH(7.0、7.5、8.0、8.5、9.0)对胚胎4个主要发育时期(受精卵期、原肠胚期、器官形成期和内骨骼形成期)耗氧率的影响.结果表明: 胚胎各个发育时期耗氧率不同,随着发育的进程而增大,受精卵期为0.082 mg·(100 eggs)^-1·h^-1,而到原肠胚期的耗氧率显著升高,为0.279 mg·(100 eggs)^-1·h^-1,到孵化期时,耗氧率达到1.367 mg·(100 eggs)^-1·h^-1;盐度对器官形成期和内骨骼形成期的耗氧率均有显著影响(P<0.05),对受精卵期和原肠胚期影响不显著(P>0.05),当盐度为30时,4个发育时期耗氧率均达到最大值,分别为0.082、0.200、0.768和1.301 mg·(100 eggs)^-1·h^-1;温度对原肠胚期、器官形成期和内骨骼形成期的耗氧率有显著影响(P<0.05),对受精卵期无显著性影响(P>0.05),在27 ℃时,胚胎4个发育时期均达到最大值,分别为0.082、0.286、0.806和1.338 mg·(100 eggs)^-1·h^-1;而pH对4个发育时期的耗氧率均无显著性影响(P>0.05),受精卵期在pH 8.0时达到最大值,为0.116 mg·(100 eggs)^-1·h^-1,原肠胚期、器官形成期、内骨骼形成期在pH 8.5时达到最大值,分别为0.281 、0.799和1.130 mg·(100 eggs)^-1·h^-1.     

Graft copolymerization of acrylic acid onto guar gum initiated by vanadium (V)–mercaptosuccinic acid redox pair

Guar gum has been modified by graft copolymerization with acrylic acid in aqueous medium using vanadium (V)–mercaptosuccinic acid redox system. The optimum reaction conditions affording maximum grafting ratio, efficiency, add on and conversion have been determined. The grafting parameters have been found to increase with increase in vanadium (V) concentration upto 1.0 × 10⁻² mol dm⁻³, but these parameters decrease on further increasing the vanadium (V) concentration. On increasing the mercaptosuccinic acid concentration from 1.0 × 10⁻² to 4.0 × 10⁻² mol dm⁻³ grafting ratio, efficiency and add on increase up to 2.0 × 10⁻² mol dm⁻³ but decrease with further increase in mercaptosuccinic acid concentration. On varying the acrylic acid concentration from 5.0 × 10⁻² to 30.0 × 10⁻² mol dm⁻³, maximum grafting ratio, efficiency and add on have been obtained at 20.0 × 10⁻² mol dm⁻³. The grafting ratio, add on and conversion increase, on increasing the H⁺ ion concentration from 1.5 × 10⁻¹ to 6.0 × 10⁻¹ mol dm⁻³. On increasing the guar gum concentration the grafting parameters increase. The grafting ratio, add on and conversion have been found to increase with time period while efficiency started decreasing after 120 min. It has been observed that %G increases on increasing the temperature up to 35 °C. The graft copolymer has been characterized by IR spectroscopy and thermogravimetric analysis.     

Use of the Hydroxamic Acid Reaction for Determining Pectinesterase Activity

The technique is: React pectinesterase contained on filter paper discs on a pectin-agar plate, flood the plate with a solution of hydroxylamine hydrochloride and then a solution of sodium hydroxide, acidify with hydrochloric acid, and then add a ferric chloride solution. The areas of substrate, desterified by the enzyme, appear as clear zones on a red background. The formation of the insoluble, red-colored ferric-pectin hydroxamic acid is characteristic of the presence of pectin carbomethoxy (methyl ester). Areas which are not colored are indicative of pectinesterase activity and this test can be used for qualitative and semi-quantitative test for approximately 1 × 10^-3 to 1 × 10^-4 pectinesterase units.     

虎斑乌贼的胚胎耗氧率

为了探究虎斑乌贼胚胎不同发育时期的耗氧率变化和几种生态因子对胚胎发育过程耗氧率的影响,试验采用封闭静水装置,对不同发育时期(12期)的耗氧率进行测定,并研究不同盐度(21、24、27、30、33)、温度(18、21、24、27、30 ℃)和pH(7.0、7.5、8.0、8.5、9.0)对胚胎4个主要发育时期(受精卵期、原肠胚期、器官形成期和内骨骼形成期)耗氧率的影响.结果表明: 胚胎各个发育时期耗氧率不同,随着发育的进程而增大,受精卵期为0.082 mg·(100 eggs)^-1·h^-1,而到原肠胚期的耗氧率显著升高,为0.279 mg·(100 eggs)^-1·h^-1,到孵化期时,耗氧率达到1.367 mg·(100 eggs)^-1·h^-1;盐度对器官形成期和内骨骼形成期的耗氧率均有显著影响(P<0.05),对受精卵期和原肠胚期影响不显著(P>0.05),当盐度为30时,4个发育时期耗氧率均达到最大值,分别为0.082、0.200、0.768和1.301 mg·(100 eggs)^-1·h^-1;温度对原肠胚期、器官形成期和内骨骼形成期的耗氧率有显著影响(P<0.05),对受精卵期无显著性影响(P>0.05),在27 ℃时,胚胎4个发育时期均达到最大值,分别为0.082、0.286、0.806和1.338 mg·(100 eggs)^-1·h^-1;而pH对4个发育时期的耗氧率均无显著性影响(P>0.05),受精卵期在pH 8.0时达到最大值,为0.116 mg·(100 eggs)^-1·h^-1,原肠胚期、器官形成期、内骨骼形成期在pH 8.5时达到最大值,分别为0.281 、0.799和1.130 mg·(100 eggs)^-1·h^-1.     

凤眼莲根分泌物氨基酸组分对根际肠杆菌属F2细菌的趋化作用

凤眼莲(Eichhornia crassipes)的根分泌物中含有Met等多种氨基酸,其中Met、GABA、Gly、Ala、Asp、Ser、Val和Leu(10^-7～10^-2mol·L^-1)均对凤眼莲的根际肠杆菌属F₂(Enterobacter sp.F₂)细菌有强烈的正趋化作用;Glu、Thr和His(10^-7～10^-3mol·L^-1)也对该菌有一定的正趋化作用;而Lys、Cys、Arg、Tyr、Pro、Asn、Gln、Ile、Phe和Typ则对该菌表现出一定的负趋化作用.对细菌的正趋化作用存在一个趋化物的最适浓度范围.具有正趋化作用的氨基酸在凤眼莲根际的浓度都较高,而具有负趋化作用的浓度则较低,这正是凤眼莲与该根际细菌结合为根际微生态系统的原因之一.     

Kinetic Behavior of Native Alkaline Phosphatase from Human Placenta

The kinetic behavior of human placental alkaline phosphatase, which catalyses the hydrolysis of p-nitrophenyl and of o-carboxyphenyl phosphates, was studied by means of graphical and non-linear regression statistical fitting analysis of data of rate versus substrate concentration. Non linear Lineweaver-Burk and Eadie-Hofstee plots and rational functions of degree 2:2 (F-test assessing the goodness of fit) show non-Michaelian kinetic behavior. In the same way, the behavior of the enzyme was also non-Michaelian in the simultaneous presence of these two substrates.

Norlaudanosoline is a key intermediate in the biosynthesis of the benzylisoquinoline alkaloids providing the benzyl-isoquinoline portion of the morphinan skeleton. This study examines a coupled reaction system for the production of norlaudanosoline from dopamine. In this coupled system, dopamine is enzymatically converted by monoamine oxidase (MAO) to 3,4-dihydroxyphenylacetaldehyde (dopaldehyde). In the presence of dopamine, this aldehyde undergoes a spontaneous Pictet-Spengler condensation to form norlaudanosoline. Three potential sources of MAO were investigated: a fungal source (Aspergillus niger), a bacterial source (Sarcina lutea) and a commercial source isolated from bovine plasma. Kinetic studies with dopamine as the substrate gave Michaelis constants (K_m) of 1.81 × 10^-5 M, 6.94 × 10^-3, and 1.61 × 10^-3 M for A. niger, S. lutea and bovine plasma oxidase, respectively. The reaction system is complicated because of the effect of the condensation reaction, so a more rigorous model was developed to account for this effect. The model was suitable for showing the effect of dopamine concentration on norlaudanosoline production alghough there were some model inadequacies. Using the model a forward rate constant for the Pictet-Spengler condensation was determined to be 6.8 × 10^-2 M^-1 s^-1 and the reverse reaction appears to be negligible. Overall conversion was 14% which is 20 times that achieved in an in situ reaction system using whole cells of Aspergillus niger.     

Redox reactions of the urate radical/urate couple with the superoxide radical anion, the tryptophan neutral radical and selected flavonoids in neutral aqueous solutions

The kinetics of several processes involving the potential antioxidant role of urate in physiological systems have been investigated by pulse radiolysis. While the monoanionic urate radical, ^·UH^-, can be produced directly by oxidation with ^·Br^-₂ or ^·OH, it can also be generated by oxidation with the neutral tryptophan radical, ^·Trp, with a rate constant of 2 × 10⁷ M^-1s^-1. This radical, ^·UH^-, reacts with ^·O^-₂ with a rate constant of 8 × 10⁸ M^-1s^-1. Also, ^·UH^- is reduced by flavonoids, quercetin and rutin in CTAB micelles at rate constants of 6 × 10⁶ M^-1s^-1 and 1 × 10⁶ M^-1s^-1, respectively. These results can be of value by providing reference data useful in further investigation of the antioxidant character of urate in more complex biological systems.     

Effect of reaction temperature and reaction time on the preparation of low-molecular-weight chitosan using phosphoric acid

Low-molecular-weight chitosan were prepared using 85% phosphoric acid at different reaction temperatures and reaction time. At room temperature, the viscosity average-molecular weights (M_v) of chitosan decreased to 7.1×10⁴ from 21.4×10⁴ after 35 days treatment. The degradation rate decreased with increasing hydrolysis time. The yields of chitosan also continuously decreased from 68.4 to 40.2% after 35 days. At 40, 60 and 80 °C, the molecular weight decreased to 3.70×10⁴, 3.50×10⁴ and 2.00×10⁴ on 8 h hydrolysis, respectively. The yields of chitosan remain at a high level compared with that at room temperature and were 86.5, 71.4 and 61.3% at 40, 60 and 80 °C treatment, respectively. The different reaction time gave chitosan with different molecular weights. At 60 °C, the molecular weight of products decreased to 7.40×10⁴ from 21.4×10⁴ within 4 h, then decreased slowly to 1.90×10⁴ in 15 h. It was also found that the water-solubility of chitosan increased as the molecular weight decreased. Results show the changes in yields and molecular weight of chitooligomers were strongly dependent on the reaction temperature and reaction time.     

Catalytic Antibodies with Perhydrolytic Activity

Monoclonal antibodies catalyzing lysis of 4-nitrophenyl esters have been created using a phosphonate as hapten in the immunization. Among 960 hybridomas screened, 3 were found to produce antibodies catalyzing hydrolysis of 4-nitrophenyl butanoate (1). Two of the antibodies accelerate the reaction by factors of 1.3 × 10⁴ and 1.1 × 10⁴, respectively, while the third antibody is significantly less effective. The two catalytically most effective antibodies also catalyze perhydrolysis of 1, i.e., lysis with hydrogen peroxide, to generate peroxybutanoic acid. Perhydrolysis was found to be the predominant reaction even in dilute solutions of hydrogen peroxide. Both antibodies also catalyze hydrolysis of both 4-nitrophenyl hexanoate and decanoate, but do not catalyze hydrolysis of 4-nitrophenyl acetate. The antibodies are more selective with respect to the aromatic part of the substrate as they do not catalyze hydrolysis of 2-nitrophenyl butanoate or 4-sulfophenyl nonanoate. Furthermore, neither of the antibodies catalyze hydrolysis of pre-formed peroxybutanoic acid.     

Sorbitol and Gluconate Production in a Hollow Fibre Membrane Reactor by Immobilized Zymomonas Mobilis

This study describes the results of a hollow fibre membrane reactor with immobilized treated cells of Zymomonas mobilis which produced sorbitol and gluconic acid continuously from fructose and glucose respectively. A productivity of 10-20 g sorbitol · L^-1 · h^-1 and 10-20 gluconate · L^-1 · h^-1 (based on total bioreactor volume) from a feed of 100 g · L^-1 each of glucose and fructose was possible at high dilution rates. Kinetic parameters describing the reaction rate of treated cells in batch reactors were used to analyse the performance of the hollow fibre membrane reactor employing significant convective mass transfer. No significant mass transfer limitation was apparent.     

Binding Specificity and Affinity of Acriflavine for Nucleic Acids

A set of conditions was developed for the specific binding of acriflavine to the DNA of intact squamous cells. This was achieved through a series of studies into the relative affinities for dye between DNA and various biopolymers by an agar gel diffusion technique. Specificity was ascertained by DNase and RNase treatment of the cells. The final conditions, based on an estimated DNA-to-dye ratio of 4:1, required a constant cell count of 100,000 and dye at a concentration of 0.0025 μg per ml in 10 ml of phosphate buffer, pH 6.0-7.4. These quantities were dictated by the sensitivity limitations of the analytical apparatus. To make use of standard fluorometric instrumentation, the whole cell population method for determining average values was followed. Free dye was analyzed after cell samples were stained, and the difference between this value and that of an aliquot of working dye was taken as the amount of bound dye. To ensure cell-free residues of dye, cells were removed by centrifugation through Teflon membrane filters. The average amount of dye which was bound to the DNA of normal squamous cells was 1.6 × 10^-7 μg (0.7 × 10^-15 mole) per cell, and the amount bound to HeLa cells was 2.3 × 10^-7 μg (1 × 10^-15 mole). These values were highly replicable, making it possible to use them as an expression of the DNA content of the cells. The mildness of the staining conditions, and the preservation of cellular integrity in this technique may permit quantitative measurements of the DNA content of living cells.     

Mechanism of reaction of nitrogen dioxide radical with hydroxycinnamic acid derivatives: A pulse radiolysis study

Nitrogen dioxide radical (NO^·₂) is known as a toxic agent produced in the metabolism of nitrates and nitrites. By the use of the pulse radiolysis technique, the mechanism of the reaction of NO^·₂ radical with hydroxycinnamic acid derivatives (HCA) was studied and the rate constants have been measured. The rate constants were found to be 7.4 × 10⁸, 7.2 × 10⁸, 8.6 × 10⁸ dm³ mol^-1s^-1 for ferulic acid, sinapic acid and caffeic acid, respectively. The reactions produce the corresponding phenoxyl radical.     

Effect of acyl donor chain length and sugar/acyl donor molar ratio on enzymatic synthesis of fatty acid fructose esters

Lipase-catalyzed synthesis of fatty acid sugar esters through direct esterification was performed in 2-methyl 2-butanol as solvent. Fructose and saturated fatty acids were used as substrates and the reaction was catalyzed by immobilized Candida antarctica lipase. The effect of the initial fructose/acyl donor molar ratio and the carbon-chain length of the acyl donor as well as their reciprocal interactions on the reaction performance were investigated. For this purpose, an experimental design taking into account variations of the molar ratio (from 1:1 to 1:5) and the carbon-chain length of the fatty acid (from C8 to C18) was employed. Statistical analysis of the data indicated that the two factors as well as their interactions had significant effects on the sugar esters synthesis. The obtained results showed that whatever the molar ratio used, the highest concentration (73 g l⁻¹), fructose and fatty acid conversion yields (100% and 80%, respectively) and initial reaction rate (40 g l⁻¹ h⁻¹) were reached when using the C18 fatty acid as acyl donor. Low molar ratios gave the best fatty acid conversion yields and initial reaction rates, whereas the best total sugar ester concentrations and fructose conversion yields were obtained for high molar ratios.     

Efficacy of the Entomopathogenic Fungus, Culicinomyces clavisporus against Larvae of the Biting Midge, Culicoides nubeculosus (Diptera: Ceratopogonidae)

Culicinomyces clavisporus, a fungal pathogen of a wide range of mosquito species, was investigated in relation to potential pathogenicity against Culicoides nubeculosus biting midge larvae. Seven different C. clavisporus strains were assayed. Each showed some degree of activity against C. nubeculosus larvae with LC₅₀ values of between 3.2×10^-5 and 1.1×10^-6 spores/mL; these effects occurred in dose-dependent manners and tended to be delayed until 72-96 h post treatment. The results are discussed in relation to incorporation of C. clavisporus into biocontrol programmes for Culicoides spp.