Responses of the ticksAmblyomma hebraeum andA. variegatum to known or potential components of the aggregation-attachment pheromone. IV. Attachment stimulation of nymphs

Ten known or potential components of the aggregation-attachment pheromone (AAP) of the ticksAmblyomma hebraeum andA. variegatum, as well as a mixture of these components and extracts of prefed males of the two species, were tested as attachment stimulants for nymphs. Unfed nymphs were confined in linen bags on the ears of rabbits that had been treated with the test compounds, mixture or extracts; the numbers attached were recorded after 24 h. InA. hebraeum, attachment was induced by four compounds (2-methyl propanoic acid, methyl salicylate,o-nitrophenol and salicylaldehyde), the mixture and extracts from both species. InA. variegatum, attachment was induced by three compounds (methyl salicylate,o-nitrophenol and salicylaldehyde), the mixture and extracts from both species. Methyl salicylate ando-nitrophenol are primary components of the AAP of bothA. hebraeum andA. variegatum. 2-methyl propanoic acid is a species-specific attachment stimulant forA. hebraeum. Salicylaldehyde, a phenolic compound, is not a naturally occurring AAP component. Nymphs of both species respond to fewer attachment stimulants than the adults and, as shown by their respective host ranges, are less dependent on the AAP in the regulation of attachment than the adults.     

The purification and spectral properties of polyphenol oxidase I fromNicotiana tabacum

Polyphenol oxidase plays a key role in plant defense systems. We report the first-time purification of polyphenol oxidase (PPO 1.14.18.1) from fresh leaves of tobacco (Nicotiana tabacum) using acetone powder, ammonium sulfate precipitation, and column chromatography with DEAE-Sephadex A-50, CM-Sephadex C-50, and Sephadex G-75. PPO I was purified approximately 71-fold (3200 U/mg). The MALDI-TOF-MS spectrum showed that the enzyme was purified to a pure protein with a molecular weight of 35700 Da. The optimum pH of PPO I was 7, the optimum temperature was 40°C, and the Km value was 6.8 mM using catechol as the substrate at pH 6.5 and with 0.05 M H₃PO₄−NaOH buffer. The maximum emission peak of PPO I was 339 nm with 16 nm of blue-shifted compared with 355 nm of free tryptophan. The UV/VIS spectra and the absence of an EPR signal are indicative of type-3 coppers, but not type-1 or type-2 coppers. PPO I and mushroom PPO have the same active center for a pair of coupled antiferromagnetic copper ions.     

Red clover coumarate 3′-hydroxylase (CYP98A44) is capable of hydroxylating p-coumaroyl-shikimate but not p-coumaroyl-malate: implications for the biosynthesis of phaselic acid

Red clover (Trifolium pratense) leaves accumulate several μmol of phaselic acid [2-O-caffeoyl-l-malate] per gram fresh weight. Post-harvest oxidation of such o-diphenols to o-quinones by endogenous polyphenol oxidases (PPO) prevents breakdown of forage protein during storage. Forages like alfalfa (Medicago sativa) lack both foliar PPO activity and o-diphenols. Consequently, breakdown of their protein upon harvest and storage results in economic losses and release of excess nitrogen into the environment. Understanding how red clover synthesizes o-diphenols such as phaselic acid will help in the development of forages utilizing this natural system of protein protection. We have proposed biosynthetic pathways in red clover for phaselic acid that involve a specific hydroxycinnamoyl-CoA:malate hydroxycinnamoyl transferase. It is unclear whether the transfer reaction to malate to form phaselic acid involves caffeic acid or p-coumaric acid and subsequent hydroxylation of the resulting p-coumaroyl-malate. The latter would require a coumarate 3′-hydroxylase (C3′H) capable of hydroxylating p-coumaroyl-malate, an activity not previously described. Here, a cytochrome P450 C3′H (CYP98A44) was identified and its gene cloned from red clover. CYP98A44 shares 96 and 79% amino acid identity with Medicago truncatula and Arabidopsis thaliana C3′H proteins that are capable of hydroxylating p-coumaroyl-shikimate and have been implicated in monolignol biosynthesis. CYP98A44 mRNA is expressed in stems and flowers and to a lesser extent in leaves. Immune serum raised against CYP98A44 recognizes a membrane-associated protein in red clover stems and leaves and cross-reacts with C3′H proteins from other species. CYP98A44 expressed in Saccharomyces cerevisiae is capable of hydroxylating p-coumaroyl-shikimate, but not p-coumaroyl-malate. This finding indicates that in red clover, phaselic acid is likely formed by transfer of a caffeoyl moiety to malic acid, although the existence of a second C3′H capable of hydroxylating p-coumaroyl-malate cannot be definitively ruled out.     

o- and p-(β-d-Glucopyranosyloxy)benzylamine from Buckwheat (Fagopyrum esculentum Moench)

Two O-glucosides (I and II) have been isolated from buckwheat seeds by column chromatography on ion-exchange resins and cellulose powder. Hydrolysis with hydrochloric acid and β-glucosidase, and spectroscopic characterization revealed their structures to be o- and p-(β-glucopyranosyloxy) benzylamine. A salicylidene derivative of II was also prepared and characterized by spectral data. This is the first report of the occurrence of these two compounds in nature.     

The polyphenol oxidase gene family in poplar: phylogeny,differential expression and identification of a novel,vacuolar isoform

Polyphenol oxidases (PPOs) are oxidative enzymes that convert monophenols and o-diphenols to o-quinones using molecular oxygen. The quinone products are highly reactive following tissue damage and can interact with cellular constituents and cause oxidative browning and cross-linking. The induction of PPO in some plants as a result of wounding, herbivore attack, or pathogen infection has implicated them in defense. However, PPO-like enzymes that act as specific hydroxylases, for example in lignan and pigment biosynthesis, have also been discovered. Here, we present the first genome-enabled analysis of a PPO gene family. The Populus trichocarpa genome was found to contain a minimum of nine complete PPO genes, and seven of these were characterized further. The PPO gene family includes both recently duplicated and divergent sequences that are 36–98% identical at the amino acid level. Gene expression profiling in poplar tissues and organs revealed that the PPO genes are all differentially expressed during normal development, but that only a small subset of PPO genes are significantly upregulated by wounding, methyl jasmonate or pathogen infection. Our studies also identified PtrPPO13, a novel PPO gene that is predicted to encode an N-terminal signal peptide. Transient expression of green fluorescent protein fusions demonstrated its localization to the vacuolar lumen. Together, our findings show that the poplar PPO family is diverse and is likely linked to diverse physiological functions.     

Location and Catalytic Characteristics of a Multipotent Bacterial Polyphenol Oxidase

The melanogenic marine bacterium Marinomonas mediterranea contains a multipotent polyphenol oxidase (PPO) able to oxidize substrates characteristic for tyrosinase and laccase. Thus, this enzyme shows tyrosine hydroxylase activity and it catalyzes the oxidation of a wide variety of o-diphenol as well as o-methoxy-activated phenols. The study of its sensitivity to different inhibitors also revealed intermediate features between laccase and tyrosinase. It is similar to tyrosinases in its sensitivity to tropolone, but it resembles laccases in its resistance to cinnamic acid and phenylthiourea, and in its sensitivity to fluoride anion. This enzyme is mostly membrane-bound and can be solubilized either by non-ionic detergent or lipase treatments of the membrane. The expression of this enzymatic activity is growth-phase regulated, reaching a maximum in the stationary phase of bacterial growth, but l -tyrosine, Cu(II) ions, or 2,5-xylidine do not induce it. This enzyme can be separated from a second PPO form by gel permeation chromatography. The second PPO is located in the soluble fraction and shows a sodium dodecyl sulfate (SDS)-activated action on the characteristic substrates for tyrosinase, l -tyrosine, and l -dopa, but it does not show activity towards laccase-specific substrates. The involvement of the multipotent PPO in melanogenesis and its relationship with the SDS-activated form and with the alternative functions proposed for multicopper oxidases in other microorganisms are discussed.     

Oxidation of phenolic and non-phenolic substrates by the lignin peroxidase of Streptomyces viridosporus T7A

Summary Numerous single-ring, aromatic, phenolic and non-phenolic compounds were tested as substrates of Streptomyces viridosporus T7A extracellular lignin peroxidase. Oxidations were monitored by spectroscopy, with and without 4-aminoantipyrine (4-AAP) as a color-forming reagent. The oxidation of phenols containing one or no carbon groups in the para position resulted in coupling with 4-AAP to form a red color. Thin layer chromatography and mass spectroscopy showed that the oxidation of vanillic acid (4-hydroxy-3-methoxybenzoic acid) and syringic acid (4-hydroxy-3,5-dimethoxybenzoic acid) resulted in a direct coupling between 4-AAP and the phenol ring to form a quinone structure. In the reaction with vanillyl acetone (4-(4-hydroxy-3-methoxyphenyl)-3-buten-2-one) and 4-AAP, 4-AAP coupled to Á-carbon of vanillyl acetone. As shown by UV-visible spectroscopy, S. viridosporus T7A peroxidase oxidized phenolic compounds, but was unable to oxidize non-phenolic ones.Paper no. 91 517 of the Idaho Agricultural Experiment Station Correspondence to: D. L. Crawford     

Isolation and characterization of the polyphenoloxidase from senescent leaves of black poplar

The polyphenoloxidase (PPO) from black poplar senescent leaves has been purified to almost complete homogeneity by a combination of ammonium sulphate precipitation, Sephadex G75 filtration and DEAE-cellulose chromatography. The purified enzyme has a MW of 60 000 and is probably a Cu⁺ enzyme. Peroxidase (PO) activity co-purifies with PPO and has the same MW as it. The two enzymes differ in pH optimum and in response to the effect of ionic strength. Natural phenols are either substrates, inhibitors or activators of black poplar PPO. This enzyme is an o-diphenoloxidase which binds substrates with K_m in the millimolar range. With caffeic and chlorogenic acids inhibition by excess substrate is observed. Benzoic acid phenols and cinnamic acid phenols are either competitive or non-competitive inhibitors of PPO. Hydroquinone is a highly potent non-competitive inhibitor of the enzyme (K_i  90 μM). Ferulic acid is a potent activator of the PPO-catalysed oxidation of catechol (K_a  0.34 mM, ν_sat/ν_o  7.7).     

Characterisation of phenol oxidase and peroxidase from maize silk

Silk of some maize genotypes contains a high level of phenolics that undergo enzymatic oxidation to form quinones, which condense among themselves or with proteins to form brown pigments. Two phenolic oxidizing enzymes, peroxidase (POD; EC 1.11.1.7) and polyphenol oxidase (PPO; EC 1.10.3.1), from maize (Zea mays L.) silk were characterised with respect to their preferred substrate, different isoforms and specific effectors. One browning silk sample with high, and two non‐browning samples with low phenolic content were investigated. Although POD oxidizes a wide range of phenolic substrates in vitro, its activity rate was independent of silk phenolic content. PPO activity, detected with o‐diphenolic substrates, was abundant only in browning silk, and low or absent in non‐browning silk. Pollination increased POD but not PPO activity. Isoelectric‐focusing (IEF) and specific staining for POD and PPO showed a high degree of polymorphism that varied with silk origin. The IEF pattern of POD revealed a number of anionic and several cationic isoenzymes, with the most pronounced having neutral pI 7 and a basic isoform with pI 10. Detected isoforms of PPO were anionic, except for one neutral form found only in browning silk, and occupied positions different from those of POD. Different inhibitory effects of NaN₃, EDTA, KCN, and L‐cysteine, as well as different impacts of a variety of cations on the oxidation of chlorogenic acid, mediated by PPO or POD, were detected. The findings are discussed in terms of a possible roles of these enzymes in defence and pollination.     

Development of an aminotransferase-driven biocatalytic cascade for deracemization of d,l-phosphinothricin

2-oxo-4-[(hydroxy)(methyl)phosphinoyl]butyric acid (PPO) is the essential precursor keto acid for the asymmetric biosynthesis of herbicide l -phosphinothricin (l -PPT). Developing a biocatalytic cascade for PPO production with high efficiency and low cost is highly desired. Herein, a d -amino acid aminotransferase from Bacillus sp. YM-1 (Ym DAAT) with high activity (48.95 U/mg) and affinity (K_m = 27.49 mM) toward d -PPT was evaluated. To circumvent the inhibition of by-product d -glutamate (d -Glu), an amino acceptor (α-ketoglutarate) regeneration cascade was constructed as a recombinant Escherichia coli (E. coli D), by coupling Ym d -AAT, d -aspartate oxidase from Thermomyces dupontii (TdDDO) and catalase from Geobacillus sp. CHB1. Moreover, the regulation of the ribosome binding site was employed to overcome the limiting step of expression toxic protein TdDDO in E. coli BL21(DE3). The aminotransferase-driven whole-cell biocatalytic cascade (E. coli D) showed superior catalytic efficiency for the synthesis of PPO from d ,l -phosphinothricin (d ,l -PPT). It revealed the production of PPO exhibited high space–time yield (2.59 g L⁻¹ h⁻¹) with complete conversion of d -PPT to PPO at high substrate concentration (600 mM d ,l -PPT) in 1.5 L reaction system. This study first provides the synthesis of PPO from d ,l -PPT employing an aminotransferase-driven biocatalytic cascade.     

Foliar oxidases as mediators of the rapidly induced resistance of mountain birch against <Emphasis Type="Italic">Epirrita autumnata</Emphasis>

Induced resistance of the mountain birch against its main defoliator Epirrita autumnata is a well-characterized phenomenon. The causal mechanism for this induced deterioration, however, has not been unequivocally explained, and no individual compound or group of traditional defensive compounds has been shown to explain the phenomenon. Phenolic compounds are the main secondary metabolites in mountain birch leaves, and the biological activity of phenolics usually depends on their oxidation. In this study, we found that the activity of polyphenoloxidases (PPOs), enzymes that oxidize o-diphenols to o-diquinones, was induced in trees with introduced larvae, and bioassays showed that both growth and consumption rates of larvae were reduced in damaged trees. PPO activity was negatively associated with both larval growth and consumption rates in trees with bagged larvae, but not in control trees. Our results suggest that the oxidation of phenolics by PPOs may be a causal explanation for the rapidly induced resistance of mountain birch against E. autumnata. This finding also helps to explain why correlations between insect performance and phenolics (without measuring indices explaining their oxidation) may not produce consistent results.     

Colorimetric Determination of Biphenyl Based on the Janovsky Reaction

The quantitatively nitrated product of biphenyl with potassium nitrate and sulfuic acid shows a characteristic red-purple color on reaction with isobutyl alcohol, acetone and alkali; this is the Janovsky reaction. The color reaction was sensitive, and the absorbance at 550 nm obeyed Beer’s law at biphenyl concentrations between 2 and 40 μg 3.5 ml of the reaction mixture. A procedure suitable for routine use is proposed.

The nitro-compound derived from biphenyl was identified as 2,2′,4,4′-tetranitrobiphenyl by Rf on TLC, as well as by mixed melting point, IR and mass spectroscopy.     

Changes in caffeic acid derivatives in sweet potato (Ipomoea batatas L.) during cooking and processing

There was an obvious decrease in caffeic acid derivatives during the boiling of cube-shaped blocks of sweet potatoes. They also decreased in a mixture of freeze-dried sweet-potato powder and water maintained at room temperature. Ascorbic acid prevented the decrease, supporting the occurrence of an enzyme reaction with polyphenol oxidase (PPO). 5-O-Caffeoylquinic acid (5-CQA, "3-O-caffeoylquinic acid" as a trivial name) and 3,5-di-O-caffeoylquinic acid (3,5-CQA), major phenolic compounds of sweet potato, did not change when they were separately heated in boiling water. When the mixture of powdered sweet potato and water was heated at 100 degrees C, there was only a negligible decrease in the total amount of phenolic compounds, and portions of 5-CQA and 3,5-CQA were found to be isomerized to 3-CQA, 4-CQA, 3,4-CQA, and 4,5-CQA. The content and composition of the phenolic compounds in sweet potatoes differed between fresh and long-stored ones, as did their response to heating.     

The assay of β-galactosidase in soil

The method for the assay of β-galactosidase introduced by Lederberg (1950) was modified and used for studies of the enzyme in the soil.o-Nitrophenyl-β-d-galactoside served as substrate. Trismaleinate buffer was found to be more suitable than phosphate buffer in the assay. The enzyme reaction was stopped by adding sulphuric acid and the incubation mixture was alkalized with sodium carbonate so that the yellow colour ofo-nitrophenol could develop. The method is sensitive and specific and requires small quantities of soil and a short incubation time.     

Comparison of sampling tabanids (Diptera: Tabanidae) by four different potential attractants

Synthetic and natural attractants in traps are used in many parts of the world to attract female tabanids. Certain attractants in different geographic regions may be ineffective or effective under different environmental conditions for horseflies. One‐octen‐3‐ol, as a compound present in bovine emanations, has a behavioural effect on many horsefly species and together with other phenolic compounds makes very effective attractant for this group of insects. As the attractiveness of the mixture of three chemicals (1‐octen‐3‐ol, acetone and ammonia solution in the proportions 5 : 3 : 2), aged donkey urine, lactic acid and fresh human urine is not yet known, it was studied in Eastern Croatia. The combination of those three chemicals and efficiency of natural attractants offers promising results. Tabanus was the most represented genus with 83% of the total collected tabanids. The chi‐squared analyses of the trapping data for canopy traps revealed that each of the attractants (mixture of three chemicals, aged donkey urine, lactic acid and fresh human urine) significantly increased the number of collected horseflies in comparison to those collected in unbaited canopy traps. Some species differences in relative response to different attractants were noted. Significantly, more specimens of Haematopota pluvialis were collected from canopy traps baited with the mixture of three chemicals when compared with traps baited with other attractants. Canopy traps baited with aged donkey urine collected significantly more Atylotus loewianus females than did traps baited with the mixture. The F‐test analysis of the trapping data for the genus Tabanus showed that there is significant difference between average number of collected specimens between mixture of three chemicals and other used attractants (lactic acid and human urine) except aged donkey urine. Finally, traps baited with the mixture of three chemicals (1‐octen‐3‐ol, acetone and ammonia solution) collected 14.5 times more tabanids than unbaited traps, whereas aged donkey urine, lactic acid, and fresh human urine‐baited traps collected 12, 3.9 and 2.5 times as many tabanids, respectively, than did unbaited traps. The mixture of three chemicals (1‐octen‐3‐ol, acetone and ammonia solution) and aged donkey urine appear to be very effective attractants for tabanids.     

Isolation and Identification of Lipase Activators from Saccharomycopsis lipolytica

Three types of lipase activators (α, β, γ) were isolated from the culture broth of Saccharomycopsis lipolytica using high performance liquid chromatography. Activator γ was the most active for the lipase reaction. One of them (β) was identified with a mixture of 3,5-dihydro xy-7-tetradecenoic acid and related compounds by the method of NMR and GC-MS analyses. The free carboxyl group in the compounds was essential for the activation of the lipase reaction.     

Antimicrobial Browning-Inhibitory Effect of Flavor Compounds in Seaweeds

Since ancient times, the antimicrobial properties of seaweeds have been recognized. However, antimicrobial activities of volatile compounds in seaweeds have not been explored so far. Here, essential oils from seaweeds including green, brown and red algae such as Laminaria japonica, Kjellmaniella crassifolia, Gracilaria verrucosa and Ulva pertusa were prepared by using SDE (simultaneous distillation and extraction) apparatus. Volatile compounds in the essential oils were identified as aldehydes, ketones, carboxylic acids, alcohols and hydrocarbons by comparison of GC-retention times and MS data with those of authentic specimens. Flavor compounds such as (3Z)-hexenal, (2E)-hexenal and (2E)-nonenal in some essential oils showed strong antimicrobial activities against Escherichia coli TG-1, and Erwinia carotovora. Inhibition of browning can be achieved during either of two stages, namely, oxidation reaction by tyrosinase or subsequent non-enzymatic polymerization. Tyrosinase activity was measured by monitoring absorbance at 475 nm originating from dopachrome formed from L-DOPA. Many kinds of aliphatic carboxylic acids, aldehydes and alcohols were used as inhibitors for PPO activity. The results indicated that the α,β-unsaturated carbonyl compounds strongly inhibit tyrosinase activity. When seaweeds are damaged or macerated, the α,β-unsaturated aldehydes such as (2E)-hexenal and (2E)-nonenal are biosynthesized via the corresponding (3Z)-unsaturated aldehydes from linolenic acid and arachidonic acid. The flavor compounds that are formed could be valuable as safe antimicrobial browning-inhibitory agents of edible seaweed origin.     

Formation of reactive oxygen species during one-electron reduction of noradrenochrome catalyzed by NADPH-cytochrome P-450 reductase

One-electron reduction of noradrenochrome catalyzed by NADPH-cytochrome P-450 reductase resulted primarily in the formation of o-semiquinone and, probably, also o-hydroquinone. Under aerobic conditions these reduced form(s) autoxidize, accompanied by the formation of reactive oxygen species, as revealed by continuous NADPH oxidation and oxygen consumption.

The presence of manganese-pyrophosphate complex contributed to autoxidation of the o-semiquinone during the reduction of noradrenochrome catalyzed by NADPH-cytochrome P-450 reductase, since the addition of the metal chelator diethylenetriamine pentaacetic acid (DETAPAC) resulted in a 34% inhibition of NADPH oxidation.

Oxygen in the ground state was found to be predominantly involved in the autoxidation of o-semiquinone during the reduction of noradrenochrome catalyzed by NADPH-cytocbrome P-450 reductase, since the addition of superoxide dismutase (SOD) to the incubation mixture only inhibited NADPH oxidation 13% and 6% in the absence and presence of DETAPAC, respectively.

The addition of catalase to the incubation mixture resulted in a slight increase in NADPH oxidation, both in the absence and in the presence of diethylenetriamine pentaacetic acid. However, no effect of catalase and SOD together on NADPH oxidation was observed, either in the absence or presence of DETAPAC.     

Reduction of acetone to isopropanol using producer gas fermenting microbes

Gasification‐fermentation is an emerging technology for the conversion of lignocellulosic materials into biofuels and specialty chemicals. For effective utilization of producer gas by fermenting bacteria, tar compounds produced in the gasification process are often removed by wet scrubbing techniques using acetone. In a preliminary study using biomass generated producer gas scrubbed with acetone, an accumulation of acetone and subsequent isopropanol production was observed. The effect of 2 g/L acetone concentrations in the fermentation media on growth and product distributions was studied with “Clostridium ragsdalei,” also known as Clostridium strain P11 or P11, and Clostridium carboxidivorans P7 or P7. The reduction of acetone to isopropanol was possible with “C. ragsdalei,” but not with P7. In P11 this reaction occurred rapidly when acetone was added in the acidogenic phase, but was 2.5 times slower when added in the solventogenic phase. Acetone at concentrations of 2 g/L did not affect the growth of P7, but ethanol increased by 41% and acetic acid concentrations decreased by 79%. In the fermentations using P11, growth was unaffected and ethanol concentrations increased by 55% when acetone was added in the acidogenic phase. Acetic acid concentrations increased by 19% in both the treatments where acetone was added. Our observations indicate that P11 has a secondary alcohol dehydrogenase that enables it to reduce acetone to isopropanol, while P7 lacks this enzyme. P11 offers an opportunity for biological production of isopropanol from acetone reduction in the presence of gaseous substrates (CO, CO₂, and H₂). Biotechnol. Bioeng. 2011;108: 2330–2338. © 2011 Wiley Periodicals, Inc.