Structures and pesticidal activities of derivatives of dinitrophenols. IX. Effects of substitution of dinitrophenols with C4 to C13 alpha-branched alkyl groups and of esterification on the eradication of apple mildew

The eradication of apple mildew caused by Podosphaera leucotricha (Ell. and Everh.) Salm. with 2-(C₄ to C₁₃ α-branched alkyl)-4,6-dinitrophenols and 4-(C₄ to C₁₃ α-branched alkyl)-2,6-dinitrophenols was examined. 4-(1-Ethylbutyl)- and most 4-(C₇ to C₁₂ α-branched alkyl)-2,6-dinitrophenols were significantly more active than their 2-alkyl-4,6-dinitrophenol analogues. Compounds containing 4-C₁₃ alkyls did not show significant activity. Activity generally increased as the α-alkyl branch lengthened. Methyl carbonates did not show lower activity than the parent phenols, but esterification of 4-(C₁₂ or C₁₃ α-branched alkyl)-2,6-dinitrophenols to ethyl carbonates or crotonates gave compounds with reduced activity. Methyl-, ethyl- or isopropyl-carbonates and crotonates of 4-(1-ethylhexyl)-2,6-dinitrophenol had much higher activity than the corresponding esters of the 4-(1-methylheptyl) isomer or of 2-(1-ethyl-hexyl)-4,6-dinitrophenol. Compounds containing long ester chains (C₇ or C₈) had less activity than 4-(1-ethylhexyl)-2,6-dinitrophenol. O-Methylation produced compounds with less activity than the parent 4-(1-ethylhexyl)- and 4-(1-propyl-pentyl)-2,6-dinitrophenols.     

Structures and pesticidal activities of derivatives of dinitrophenols. X. Effects of substitution od dinitrophenols with C3 to C13 alpha-branched alkyl groups and of esterification on the eradication of barley mildew

The studies of the eradication of barley mildew due to Erysiphe graminis Mérat with alkyldinitrophenols reported earlier¹ were continued. The degree of eradication was highest with 4-(C₉ α-branched alkyl)-2,6-dinitrophenols. 4-Alkyl-2,6-dinitrophenols containing a heptyl or higher alkyl branch were significantly less active, and compounds containing the C₁₂ or C₁₃ alkyls were not active since the most compact of the C₁₂ α-branched alkyls is 1-pentylheptyl. 2-(1-Methylheptyl)- and 2-(1-propylpentyl)-4,6-dinitrophenols had high activity, but their esters showed reduced activity. Esterification of 4-(α-branched alkyl)-2,6-dinitrophenols to methyl carbonates did not affect the activity shown by the compounds, but when certain 4-C₁₀ and C₁₁ alkyl-2,6-dinitrophenols were esterified to ethyl carbonates the activity of the products was reduced. Also activity was diminished by conversion of some 4-C₉ and C₁₀ alkyl-2,6-dinitrophenols to crotonates. Whereas the methyl- and ethyl- carbonates of 4-(1-ethylhexyl)-2,6-dinitrophenol gave consistently high degrees of eradication of barley mildew, the performance of the crotonate of this phenol was not consistent. Lower aliphatic esters of the active C₈-alkyl phenols were themselves active. Esterification of 4-(1-ethylhexyl)-2,6-dinitrophenol to the benzoate, isopropyl carbonate or S-methyl thiolocarbonate gave compounds with substantially reduced activity. 2-t-Butyl-4,6-dinitrophenyl or 4-t-butyl- or t-octyl-2,6-dinitrophenyl esters gave little or no significant eradication.     

Structures and pesticidal activities of derivatives of dinitrophenols. XII.—In vitro activity of C3 to C13 α-branched alkyldinitro-phenols and their esters and ethers against the spores of Venturia inaequalis and other fungi

The activity in vitro of 2-(C₃ to C₁₃ α-branched alkyl)-4,6-dinitrophenols and 4-(C₄ to C₁₃ α-branched alkyl)-2,6-dinitrophenols and of their various esters and ethers was determined against the spores of Venturia inaequalis (Cooke) Wint., Botrytis cinerea Pers., Fusarium bulbigenum, Cooke & Massee, var. lycopersici (Brushi) Wollenw. and Cercospora melonis Cooke. Several alkyldinitrophenols were highly active against Venturia and the 2-alkylphenols were generally more active than the 4-alkylphenols. The alkyldinitrophenols were not effective against the spores of Botrytis, Fusarium and Cercospora.. Esterification of several lower alkyldinitrophenols to methyl carbonates gave compounds with enhanced activity, but methyl carbonates of C₇ to C₁₁ alkyl dinitrophenols showed considerably reduced activity. Esterification to ethyl- or other alkyl-carbonates gave compounds with considerably reduced activity against Venturia. Esterification of 2-isopropyl- and 2-t-butyl-4,6-dinitrophenols to crotonates gave compounds with enhanced activity against Venturia, but crotonates of other phenols showed reduced activity. The acrylates of several C₆ to C₈ alkyl dinitrophenols proved highly active. Ethers of active phenols showed considerably reduced activity.     

Structures and pesticidal activities of derivatives of dinitrophenols. 8. Effects of substitution with C5 to C13-s-alkyl groups and of esterification on the acaricidal activity of dinitrophenols

38 methyl-, 37 ethyl- and 19 other alkyl-carbonates, 37 crotonates, 10 acrylates and 17 other esters, and 15 methyl ethers of 2-(C₅ to C₁₃-s-alkyl)-4,6-dinitro- and 4-(C₄ t4 to C₁₃-s-alkyl)-2,6-dinitrophenols were synthesised, and their activities against Tetranychus telarius (greenhouse red spider mite) were investigated. 2-s-Alkyl-4,6-dinitrophenols and esters were more active than their 4-s-alkyl-2,6-dinitro- analogues, acaricidal activity remaining high with the 4,6-dinitrophenols up to 2-(C₁₁-s-alkyl). Generally compactness of the 2-s-alkyl group aided activity. Methyl ethers had very low activity. Esters of 2-(C₃ to C₇-s-alkyl)-4,6-dinitrophenols were more acaricidal than the parent phenols, but the reverse was the case with C₈ to C₁₃-s-alkyl compounds. Crotonates and other esters were generally less active than methyl carbonates. The methyl carbonates of 2-(1-ethylhexyl)- and 2-(1-propylpentyl)-4,6-dinitrophenols were found to be of particular economic interest as acaricides.     

Structures and pesticidal activities of derivatives of dinitrophenols. I. Structure and acaricidal activity of certain carbonates of dimitrophenols

The activities against Tetranychus telarius (greenhouse red spider mite) of alkyl, aralkyl and aryl 2-substituted-4,6-dinitrophenyl and 4-substituted-2,6-dinitrophenyl carbonates have been investigated. Alkyl carbonates of 2-s-butyl-, 2-(l-methyl-n-butyl)- and 2-t-butyl-4,6-dinitrophenols were highly active. The carbonates of the first two phenols were more active than the phenols themselves. A detailed study of the structure–activity relation revealed that increase in length of the ester chain caused decrease in acaricidal activity. The branched alkyl carbonates of 2-s-butyl- and 2-(l-methyl-n-butyl)-4,6-dinitrophenol were as active as, whereas those of 2-t-butyl-4,6-dinitrophenol were less active than, the corresponding n-alkyl carbonates. The thiolo-, thiono-, and dithio-carbonates were less active than their oxy-analogues and the aralkyl and aryl carbonates less active than their alkyl analogues.     

Structure and pesticidal activities of derivative of dinitrophenols. 3. Structure and acaricidal activity of certain carbonates of 2-alkyl-5-methyl-4,6-dinitrophenols and related compounds

The activities of alkyl 2-alkyl-5-methyl-4,6-dinitrophenyl carbonates and of related compounds against Tetranychus telarius (greenhouse red spider mite) were examined. The 5-methyl substituent slightly reduced the toxicity to mites, but not to mammals. The carbonates of 2-isopropyl- and 2-s-butyl-5-methyl 4,6-dinitrophenols were more acaricidal than the parent phenols. The increase in chain length of the alkoxyl group in the carbonates caused a decrease in acaricidal activity. In contrast to the branched carbonates of 2-t-butyl-4,6-dinitrophenol their 5-methyl-analogues were as active as the normal homologues. The cyclohexyl and phenyl carbonates of the 5-methyl-analogues had better activity than those without the 5-methyl substituent. The thiolo- and dithio-carbonates were found to be less active than their oxy-analogues.     

Studies in fungitoxicity. VII.—Fungicidal activity of certain ethylenes and heterocyclic compounds substituted with the 2,4-dinitrophenylthio- group

Certain ethylenes and heterocyclic compounds substituted with the 2,4-dinitrophenylthio- group were synthesised and tested for activity against Venturia inaequalis, Botrytis cinerea, Fusarium bulbigenum and Cercospora melonis. 1-Alkylthio-1-(2,4-dinitrophenylthio)-2-cyano-2-(ethoxycarbonyl or cyano) ethylenes in which the Ph-S bond is not shielded by the alkyl substituent from attack, perhaps by nucleophilic cell constituents, were found to be active against Venturia. 3,5-Di(methyl- or ethyl-thio)-4-cyanoisothiazoles were also active against Venturia. 2-Substituted-5-(2,4-dinitrophenylthio)-1,3,4-oxadiazoles had no or low fungicidal activity, and of several 2-(2,4-dinitrophenylthio) heterocyclic compounds that were tested 2-(2,4-dinitrophenylthio)-benzothiazole and -benzoxazole had good activity against Venturia. The infra-red and proton magnetic resonance spectra of several compounds were examined.     

Alpha-Amylase from Streptomyces aureofaciens — Purification and Properties

The amylase from a chlortetracycline-producing strain of Streptomyces aureofaciens was purified by different treatments until it was found to be homogeneous. The molecular weight of the enzyme was about 40000, the p_H -optimum was in the range of 4,6–5,3 and the optimal temperature was around 40°C. The activity was not influenced by Ca-ions. The enzyme cleaved substrates in an endomechanism producing glucose, maltose and maltotriose in α-configuration as main products. It was decided to be α-amylase, 1,4-α-D -Glucan glucanohydrolase, EC 3.2.1.1.     

Structures and pesticidal activities of derivatives of dinitrophenols. VI. Structure and pre-emergence herbicidal activity of certain alkyldinitrophenols and their esters

The activities of DNOC, dinoseb, dinoterb and dinosam and the 5-methyl analogues (I; R = Bu^s, Bu^t, or CHMePrⁿ and R' = Me) of these phenols against Capsella bursa-pastoris, Chenopodium album, Senecio vulgaris, Stellaria media and Rumex spp., and their effects on peas, oats, beet, cabbage and carrots, were investigated by pre-emergence application to soil. The effects of esterification of these phenols with aliphatic acids, of esterification of dinoterb with dibasic acids, aromatic acids, substituted carbonic acids, and of etherification on activity were investigated. 2-t-Butyl phenols and their aliphatic esters proved more active than the corresponding compounds of the other phenols. Acetylation improved the activity of dinoterb and medinoterb. Halogenation reduced the activity of the acetate of dinoterb, but unsaturation of the ester chain enhanced the activity of dinoterb esters. Esterification of dinoterb with aromatic acids, substituted carbonic acids, oxalic and adipic acids, and etherification led to compounds of low activity. In general, all compounds caused little damage to peas and oats. Dinoterb esters damaged cabbage plants. Esters of medinoterb were generally less damaging to crops than were dinoterb esters.     

Printing ink compounds in foods: UK survey results

Three hundred and fifty foodstuffs packaged in printed paper/board were purchased from UK retail outlets. Solvent extracts of all foods and associated quality assurance samples were analysed by gas chromatography–mass spectrometry (GC-MS) to determine the presence and concentrations of 20 printing ink compounds: benzophenone, 4-methylbenzophenone, 2-methylbenzophenone, 3-methylbenzophenone, 4-hydroxybenzophenone, 2-hydroxybenzophenone, 4-phenylbenzophenone, methyl-2-benzoylbenzoate, 1-hydroxycyclohexyl phenyl ketone, 2-isopropylthioxanthone, 4-isopropylthioxanthone, 2,4-diethyl-9H-thioxanthen-9-one, 2,2-dimethoxy-2-phenylacetophenone, 2-methyl-4′-(methylthio)-2-morpholinopropiophenone, 4-(4-methylphenylthio)benzophenone, ethyl-4-dimethylaminobenzoate, 2-ethylhexyl-4-(dimethylamino)benzoate, N-ethyl-p-toluene-sulphonamide, triphenyl phosphate and di-(2-ethylhexyl) fumarate. The presence of one or more of the compounds benzophenone, 4-phenylbenzophenone, methyl-2-benzoylbenzoate, 1-hydroxycyclohexyl phenyl ketone, 2,2-dimethoxy-2-phenylacetophenone, 4-(4-methylphenylthio)benzophenone, ethyl-4-dimethylaminobenzoate, 2-ethylhexyl-4-dimethylaminobenzoate and triphenyl phosphate was confirmed in some food samples. Analysis of the associated packaging material was also carried out to confirm whether or not it was likely that the occurrence of these compounds in the foods was due to migration from the printed paper/board packaging. With the exception of triphenyl phosphate, detected in one foodstuff, all the packaging material contained the substance(s) found in the food.     

Untersuchung von Provolone-K?se-Aroma

Zusammenfassung Provolone, ein italienischer Käse aus Kuhmilch, wurde auf die folgenden Verbindungsklassen, die als Bestandteile von Käse-Aromen schon bekannt sind, untersucht: Fettsäuren: Die freien kurzkettigen und mittelkettigen Fettsäuren des Provolone-Käses wurden gaschromatographisch qualitativ und quantitativ bestimmt. Valerian-, Heptan- und Nonansäure wurden erstmals nachgewiesen. Mittels GC-MS warden außerdem die Anteisosäuren C₁₁, C₁₂, C₁₄, C₁₅ und C₁₇, sowie die einfach ungesättigten Säuren C₁₂, C₁₄, C₁₆ und C₁₈ identifiziert. Alkohole: Durch Gaschromatographie und Dünnschichtchromatographie der 4-Dimethylamino-3,5-dinitrobenzoate wurden acht 1-Alkanole und sechs 2-Alkanole in Aroma-Konzentraten aus Provolone-Käse nachgewiesen. GC-MS-Untersuchungen bestätigten die hier nachgewiesenen Alkohole. Carbonylverbindungen: Die Carbonylverbindungen wurden als 2,4-Dinitrophenylhydrazone (DNPHs) aus dem Aroma-Konzentrat gewonnen. Nach Abtrennung der sauren DNPHs untersuchten wir die neutralen DNPHs dünnschichtchromatographisch und die aus ihnen freigesetzten Carbonylverbindungen gasehromatographisch. Wir konnten bisher 16 Aldehyde, 4 Methylketone und Diacetyl als neutrale Carbonylverbindungen in Provolone-Käse nachweisen. Die sauren DNPHs reduzierten wir mit Zinn/Salzsäure zu den entsprechenden Aminosäuren, die in einem automatischen Aminosäuren-Analysator analysiert wurden. Wir konnten 14 -Ketosäuren als Aminosäuren im Provolone-Käse nachweisen. Ester: Durch GC-MS wurden in der Neutralfraktion des Aroma-Konzentrats 2 Methyl-, 4 Äthyl-, 4 n-Propyl-, 2 n-Butylester und 1 sec Butylester der kurzkettigen Fettsäuren nachgewiesen. Amine: Die Amine isolierten wir aus dem Entgasungskondensat nach Abtrennung der sauren und neutralen Komponenten als Aminhydrochloride. Sie wurden nach Derivatisierung dünnschichtchromatographisch und gaschromatographisch untersucht. Wir konnten bisher 8 primäre Aklylamine und 4 Dialkylamine als Aroma-Komponenten des Provolone-Käses nachweisen. Freie Aminosauren: Die freien Aminosäuren wurden aus dem Käse nach Fällung des Proteins isoliert und unter Verwendung eines inneren Standards qualitativ und quantitativ bestimmt.

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Investigation of provolone cheese flavour

Summary Provolone, an Italian cheese made from cow's milk, has been analyzed w.r.t. the following classes of compounds which are already known as components of cheese flavours: Fatty Aacids: The short chain- and medium-chain free fatty acids of Provolone cheese were determined qualitatively and quantitatively via GLC. Valerie-, heptanoic- and nonanoic acids were identified for the first time. Moreover, the anteiso-acids C₁₁, C₁₃, C₁₄ C₁₅ and C₁₇, as well as the mono-unsaturated acids C₁₂, C₁₄, C₁₆, and C₁₈ were identified by means of combined gaschromatography-mass spectrometry (GLC-MS). Alcohols: By means of gas chromatography (GLC) and thinlayer chromatography (TLC) of the 4-dimethylamino-3.5-dinitrobenzoates, eight 1-alkanols and six 2-alkanols were identified in the flavour concentrate of Provolone. GLC-MS investigations confirmed the presence of the identified alcohols. Carbonyl Compounds: The carbonyl compounds were obtained from the flavour concentrate as 2.4-dinitrophenylhydrazones (DNPHs). After separating the acidic DNPHs, we examined the neutral DNPHs via TLC and the free carbonyl compounds regained from these via GLC. Hitherto we were able to identify 16 aldehydes, 4 methyl ketones and diacetyl as neutral carbonyl compounds in Provolone cheese. We reduced the acidic DNPHs with Sn/HCl to the corresponding amino acids which were subsequently analyzed with an automatic amino acid analyzer. We were able to identify 14 -keto acids as amino acids in Provolone cheese. Esters: By means of GLC-MS of the neutral fraction of the flavour concentrate, 2 methyl-4 ethyl-, 4 n-propyl-, 2 n-butyl- and one sec-butyl ester of the short-chain fatty acids were, identified. Amines: We isolated the amines as amine hydrochlorides from the degassing condensate, after separating the acidic and neutral components. The amine hydrochlorides were then transformed into their appropriate derivatives, subsequently investigated via TLC and GLC. Hitherto we could identify 8 primary alkyl amines and 4 dialkyl amines. Free Amino Acids: The free amino acids were isolated from the cheese after precipitation of the protein. They were then analyzed qualitatively and quantitatively in an automatic amino acid analyzer using an internal standard.

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Für die sorgfältige Durchführung der Untersuchungen danken wir Frl. L. Liebhorr, Fran A. von Voss, Frl. U. Dahncke, Frl. M. Schuldt und Herrn v. d. Hoek. Herrn Dr. W. Freytag sei hier für die wertvollen Diskussionen, Herrn Dr. H. D. Pruss für die Aminosäure-Analyse und Herrn Dr. A. Zeman für die MS-Untersuchungen gedankt.     

Proteinaceous Surfactants Produced from Gelatin by Enzymatic Modification: Evaluation for Their Functionality

Proteinaceous surfactants were prepared by applying the “one-step process” which permitted covalent incorporation of amino acid esters directly into proteins during treatment with papain. Gelatin was used as a hydrophile and n-alkyl esters of L-leucine as lipophiles. Each of the hydrophile-lipophile mixtures was incubated with papain under the following conditions: medium, 1M carbonate (pH 9) or a 20:80 mixture of acetone-1M carbonate (pH 9) containing 2 mM 2-mercaptoethanol; concentration of gelatin in the medium, 33% (w/w); L-leucine ester vs gelatin ratio, 0.1 mole/100g; papain vs gelatin, 1% (w/w); incubation period, 15 min; and temperature, 37°C. The enzymatic reaction was stopped by adding 1N HCl and the product purified by dialysis followed by washing with hot acetone or dichloromethane to remove low-molecular species. Each product was found to be a mixture of peptides having a wide range of molecular weight, with an average at approximately 7,500 daltons. The amounts of the alkyl moieties covalently incorporated were in a range 1.1–1.2 moles per 7,500g of the products. Their surfactancy varied depending particularly on the carbon number of the alkyl moiety; the products resulting from the incorporation of C₄–C₆ alkyl esters of leucine showed greater whippability, whereas the incorporation of the C₁₀–C₁₂ alkyl esters gave products having a higher ability to stabilize an o/w type emulsions.     

Inhibition ofIn VitroHuman LDL Oxidation by Phenolic Antioxidants from Grapes and Wines

Current research suggests that wine contains substances that may reduce the mortality rate from coronary diseases. The oxidation of low-density lipoprotein (LDL) is thought to be a key step in the development of atherosclerosis. Phenolic fractions of a Petite Syrah wine were evaluated for their antioxidant activity in inhibiting LDL oxidation in vitro . The more active fractions contained components of the catechin family. The catechin oligomers and the procyanidin dimers (B₂, B₃, B₄, B₆, B₈) and trimers (C₁, C₂) were extracted, isolated and purified from grapes seeds. These compounds were tested for their inhibition of LDL oxidation, along with other monomeric wine phenolics. The procyanidin dimers B₂ and B₈, and trimer C₁, and the monomers catechin, epicatechin and myricetin had the highest antioxidant activity. The procyanidin dimers B₃, B₄ and C₂ and the monomers gallic acid, quercetin, caffeic acid, and rutin, and a group of compounds that included the dimer B₆, ellagic acid, sinapic acid, cyanidin had lower antioxidant activity and α-tocopherol had the least activity. Thus, the numerous phenolic compounds found in wine are potent antioxidants in inhibiting LDL oxidation in vitro .     

Purification and Characterization of the Commercialized,Cloned Bacillus megaterium α-Amylase. Part I: Purification and Hydrolytic Properties

An amylolytic enzyme, originally isolated from Bacillus megaterium. was shown to increase the maximum amount of dextrose produced during saccharification by decreasing the amount of residual oligosaccharides. The enzyme, now commercially produced in a genetically engineered strain of Bacillus subtilis, was purified to homogeneity from the commercial product. A combination of gel permeation chromatography in the presence of 1.0 M NaCl and chromatofocusing between pH 9.0 and 7.0 were used to obtain the pure enzyme. The molecular weight of the Bacillus megaterium α-amylase was 59,000 by SDS gel electrophoresis, and the isoelectric point was 8.9 to 9.0. The enzyme was shown to possess both hydrolytic and transferase activity. The enzyme hydrolyzed a wide variety of soluble substrates. The rate of hydrolysis was greatest on soluble starch; α(1,6)-branched substrates and cyclodextrins were hydrolyzed more slowly.     

PURIFICATION AND CHARACTERIZATION OF AN ENDOPEPTIDASE FROM PSEUDOMONAS FLUORESCENS ATCC 948

Intracellular, ca 55 kDa monomeric endopeptidase (PsPepO) from Pseudomonas fluorescens ATCC 948 was purified to homogeneity by chromatography on Q-Sepharose, Sephacryl 200, Phenyl-Superose and Mono Q. It was strongly inhibited by the metalloproteinase inhibitor, 1,10 phenanthroline and by dithiothreitol, but less strongly by EDTA; it was stimulated by Co²⁺. Activity was optimal at pH 7.0 and 40–45C, with considerable activity at pH 5.0 and 12C. The enzyme was relatively heat-stable with a D_80Cvalue of 1.2 min. It did not hydrolyze α_s1-, β-or κ-casein (CN) and peptides with less than 5 amino acids but readily hydrolyzed α_s1-CNfl-23, α_s1-CN f157-164, α_s1-CN f165-199 and various β-CN fragments and peptide hormones. On α_s1-CN fl-23, α_s1-CN f165-199, insulin B-chain and bradykinin, it mainly catalyzed the hydrolysis of peptide bonds in which a hydrophobic amino acid (most commonly Phe or Leu) residue occupied the P'₁position. β-CN f193- or 194-209, which are the source of bitter peptides in cheese ripening, were hydrolyzed slowly or not at all. β-CN fragments from the sequence 58-70 were degraded or did not inhibit the endopeptidase activity as well as β-CN f193-209. The characteristics of the endopeptidase of Ps. fluorescens ATCC 948 were compared with those of lactococcal endopeptidases.     

Proteinaceous Surfactants Produced from Gelatin by Enzymatic Modification: Application to Preparation of Food Items

Proteinaceous surfactants produced from gelatin by papain-catalyzed incorporation of L-leucine n-alkyl esters were used as ingredients replacing conventional surfactants for food use. The incorporation of leucine Cz-C6 alkyl esters gave surfactants effective in making snow jelly. In ice cream making, a leucine C₁₂ alkyl ester-incorporated product used as the surfactant gave a high degree of overrun even in a few minutes from the start of whipping. This surfactant was applicable also to making mayonnaise, with formation of a tine emulsion having favorable hardness and adhesiveness. In bread making as well, the same surfactant was usable and found preferable to monostearin. The use of this surfactant resulted in satisfactory loaf quality as well as slow staling of bread over a long period of storage.     

ISOLATION AND CHARACTERISATION OF THE AMYLOTIC SYSTEM OF SCHWANNIOMYCES CASTELLII

The amylolytic system of Schwanniomyces castellii has been isolated and purified by means of ultrafiltration followed by polyacrylamide gel electrophoresis. Both α-amylase and glucoamylase were purified. α-Amylase activity was stable from pH 5·5 to 6·5 and glucoamylase activity was stable at a more acidic range of pH 4·2 to 5·5. The optimal temperature of α-amylase activity was between 30 and 40°C with rapid deactivation at 70°C. The optimal temperature of glucoamylase was 40 to 50°C with rapid decline of activity at 60°C. The K_m of α-amylase with soluble starch as the substrate was 1·15 mg/ml and the K_m of glucoamylase with the same substrate was 10·31 mg/ml. Glucoamylase was able to hydrolyze α-1, 4 and α-1,6 glucosidic linkages, as demonstrated by its ability to hydrolyse maltose and isomaltose respectively, whereas α-amylase could hydrolyse α-1,4 glucosidic linkages only. α-Amylase was shown to be a glycoprotein, whereas no carbohydrates were associated with glucoamylase.     

Determination of herbicide ZJ0273 residue in rapeseed by radioisotopic tracing method

Residue of herbicide ZJ0273 (propyl 4-(2-(4,6-dimethoxypyrimidin-2-yloxy)benzylamino)benzoate) in rapeseed under simulated field conditions was evaluated by radioisotopic tracing method. Radio-HPLC and HPLC–MS analysis revealed that the parent ZJ0273 is the only radioactive residue in rapeseed. The residue of propyl 4-(2-(4,6-dimethoxypyrimidin-2-yloxy)[phenyl-U-¹⁴C₆]benzylamino)benzoate (RCZJ) in postharvest rapeseed from the rape seedlings (Brassica napus L.) in red clayey soil (S₁) and in fluvio marine yellow loamy soil (S₂) were 0.67 and 1.24 nmol/plant, respectively, based on the ¹⁴C radioactivity and the specific activity of the labelled ZJ0273. The RCZJs in the rapeseed (1 kg) from the seedlings in S₁ and in S₂ were 40.13 and 45.33 nmol, respectively, and the amounts of ZJ0273 in the rapeseed (1 kg) were 16.97 and 19.17 μg. The results suggested that only a trace level of residue of ZJ0273 presents in rapeseed due to the poor root absorption of ZJ0273 and the weak translocation of ZJ0273 from rape leaves.     

Identification and characterization of surface lipid components of the dried-bean beetle Acanthoscelides obtectus (Say) (Coleoptera: Bruchidae)

The dried-bean beetle Acanthoscelides obtectus is a major post-harvest pest, which can cause up to 30% losses from dry-stored beans. For the use of microbial agents for biocontrol of A. obtectus, it is of importance to identify cuticular lipids of this pest, if we are to understand the factors responsible for preferential adhesion or selective repulsion of entomopathogenic fungi that are potentially useful for biocontrol.The cuticular lipids of adult A. obtectus of both sexes were found to consist of hydrocarbons, aldehydes, methyl- and ethyl-esters of fatty acids, triacylglycerols, free fatty acids, alcohols and sterols. All the fatty acids identified in this study were found in both ether extracts and dichloromethane extracts. The dominant fraction of all the lipids isolated from this species consisted of C_16:0, C_18:0, C_18:1, C_18:2 and C_18:3 compounds. Males and females contained similar amounts of hexadecanoic acid, but there was a significant difference between the total amounts of C_18:0, C_18:1 and C_18:2 acids in the two groups. We have also successfully identified one of the sesquiterpenes present in the cuticle as α-farnesene.     

Preparation of [13 C 3]-melamine and 13 C 3]-cyanuric acid and their application to the analysis of melamine and cyanuric acid in meat and pet food using liquid chromatography-tandem mass spectrometry

For the determination of melamine and cyanuric acid the labelled internal standards [¹³ C ₃]-melamine and [¹³ C ₃]-cyanuric acid were synthesized using the common substrate [¹³ C ₃]-cyanuric chloride by reaction with ammonia and acidified water, respectively. Standards with excellent isotopic and chemical purities were obtained in acceptable yields. These compounds were used to develop an isotope dilution liquid chromatography/mass spectrometry (LC/MS) method to determine melamine and cyanuric acid in catfish, pork, chicken, and pet food. The method involved extraction into aqueous methanol, liquid–liquid extraction and ion exchange solid phase clean-up, with normal phase high-performance liquid chromatography (HPLC) in the so-called hydrophilic interaction mode. The method had a limit of detection (LOD) of 10 µg kg^?1 for both melamine and cyanuric acid in the four foods with a percentage coefficient of variation (CV) of less than 10%. The recovery of the method at this level was in the range of 87–110% and 96–110% for melamine and cyanuric acid, respectively.