Polyphenol Oxidase, Peroxidase and PhenylalanineAmmonium Lyase Induced in Postharvest Peach Fruitsby Inoculation with Pichia membranefaciensor Rhizopus stolonifer

Rhizopus rot of peach fruits could be significantly suppressed by Pichia membranefaciens. Polyphenol oxidase (PPO), peroxidase (POD) and phenylalanine ammonium-lyase (PAL) activities induced by inoculation with P. membrane faciens or R. stolonifer were studied in postharvest peach fruits. The activities of PPO and PAL in peaches increased significantly after being inoculated with P. membrane faciens + R. stolonifer by 24 h, the activities maintained at a high level throughout the experiment. Under the condition of infected with R. stolonifer alone, activity of PPO and PAL could also increased, but the levels were lower than those treated with P. membrane faciens+ R. stolonifer. However, fruits inoculaed with P. membrane-faciens + R. stolonifer or R. stolonifer alone did not stimulated POD activity. The results suggest that the activation of these defense enzymes is involved in the action of P. membrane faciens against R. stolonifer.

Potential of plant polyphenol oxidases in the decolorization and removal of textile and non-textile dyes

In this study an effort has been made to use plant polyphenol oxidases; potato （Solanum tuberosum） and brinjal （Solanum melongena）, for the treatment of various important dyes used in textile and other industries. The ammonium sulphate fractionated enzyme preparations were used to treat a number of dyes under various experimental conditions. Majority of the treated dyes were maximally decolorized at pH 3.0. Some of the dyes were quickly decolorized whereas others were marginally decolorized. The initial first hour was sufficient for the maximum decolorization of dyes. The rate of decolorization was quite slow on long treatment of dyes. Enhancement in the dye decolorization was noticed on increasing the concentration of enzymes. The complex mixtures of dyes were treated with both preparations of polyphenol oxidases in the buffers of varying pH values. Potato polyphenol oxidase was significantly more effective in decolorizing the dyes to higher extent as compared to the enzyme obtained from brinjal polyphenol oxidase. Decolorization of dyes and their mixtures, followed by the formation of an insoluble precipitate, which could be easily removed simply by centrifugation.

Effects of Terpinen-4-ol on Four Metabolic Enzymes and Polyphenol Oxidase (PPO) in Mythimna separta Walker

To study insecticidal mechanism of terpinen-4-ol, a main insecticidal composition in the essential oil of Sabina vulgaris, the 5th instar larvae of Mythimna separta, were investigated with terpinen-4-ol by topical application. The activities of phosphatase, glutathione S-transferase （GSTs）, cytochrome P450 （P450）, and polyphenol oxidase （PPO） of tested insects were determined in all poisoning stages, including exciting stage, convulsing stage, paralysis stage, and recover stage. The result showed that the activities of both acid phosphatase （ACP） and alkaline phosphatase （AKP） in treated insects were induced by terpinen-4-ol, but ACP was inhibited in paralysis stage. The activities of GSTs were inhibited in exciting stage, convulsing stage, and paralysis stage, but gradually recovered in recover stage. O-demethylase activity of cytochrome P450 was inhibited by terpinen-4-ol, and the inhibition rate in all poisoning stages were 26.27, 46.03, 80.24, and 90.22%, respectively. PPO activities were strongly inhibited by terpinen-4-ol both in vitro and in vivo. In conclusion, the activities of P450, GSTs, and PPO could have relation with toxicity of terpinen-4-ol against larvae of the Mythimna separta, but recover stage of the poisoning insects might be related to GSTs induced. As a new insecticide or synergist, terpinen- 4-ol has a potential value in field of insecticide resistance management.

Study on Enzymatic Kinetics of Nephelium lappaceum Polyphenol Oxidase

[Objective] This study aimed to investigate the enzymatic kinetics of polyphenol oxidase in peel of Nephelium lappaceum to explore the environmental factors affecting its catalytic activity. [ Method ] With N. lappaceum peel as the experimental material and catechol as substrate, effects of temperature, pH, four inhibitors （EDTA, Vc, sodium bisulfite and citric acid） and substrate concentration on the activity of polyphenol oxidase were investigated. [ Result] The optimal temperature for polyphenol oxidase in N. lappaceum peel was 40 %, and the optimal pH was 6.8. EDTA, Vc, sodium bisulfite and citric acid all showed ideal in- hibitory effects on the activity of polyphenol oxidase; specifically, EDTA had the strongest inhibitory effect. [ Conclusion] At low temperature, EDTA, Vc, sodi- um bisulflte and citric acid with certain concentrations can inhibit the catalytic activity of polyphenol oxidase in peel of N. lappaceum, extend the storage and trans- portation time of N. lappaceum fruit, and inhibit the browning. This study provides theoretical basis for the development and utilization of polyphenol oxidase in peel of N. lappaceum.

Immobilization and Properties of Polyphenol Oxidase

[Objectives]This study was conducted to investigate the effects of embedding conditions on activity and catalytic properties of immobilized polyphenol oxidase.[Methods]Polyphenol oxidase was immobilized in a polymer material by the embedding method,and the optimal immobilization conditions were obtained by single factor tests:CaCl_(2) concentration 2.0%,sodium alginate concentration 2.0%,immobilization time 2 h and mass ratio of enzyme to carrier 10 mg/100 g,under which the immobilized enzyme activity was 93.33 U/g.Under the above conditions,the properties of polyphenol oxidase immobilized by sodium alginate(A-PPO)and free polyphenol oxidase were studied.[Results]The thermostability of A-PPO was better than that of the free enzyme,but the pH stability of A-PPO was inhibited.The Michaelis constant K_(m) values of free polyphenol oxidase and A-PPO were 0.37 and 0.48 mmol/L,respectively,and the maximum reaction rate V_(max) values were 0.38 and 0.51 mmol/(L·g),respectively.[Conclusions]This study provides a theoretical basis for the study of the properties of polyphenol oxidase.

Kinetics of Inhibition of Polyphenol Oxidase Obtained from Tobacco Nicotiana Tobacum

In this study, the kinetics of inhibition of polyphenol oxidase by L-cysteine has been investigated. The inhibition of tobacco polyphenol oxidase was studied using the progress-of-substrate-reaction method proposed by Tsou, following the substrate reaction during irreversible inhibition of the enzyme activity. Analysis of the inhibition kinetics shows that inhibition occurs by an irreversible and non-complexation reaction. The microscopic rate constants were determined for reaction of the inhibitor both with the free enzyme and with the enzyme-substrate complex. The results show that the presence of the substrate has a significant protective effect of the enzyme against inactivation by L-cysteine.

Inhibitory Kinetics of p-Substituted Benzaldehydes on Polyphenol Oxidase from the Fifth Instar of Pieris Rapae L.

Polyphenol oxidase （PPO） is the enzyme responsible for enzymatic browning during the growth of insects. It is also involved in defense reactions and is related with immunities in insects. PPO a metalloenzyme oxidase, catalyzes the oxidation of o-diphenol to o-quinone. The present paper describes the effects of benzaldehyde and its p-substituted derivatives on the activity of PPO from the fifth instar of Pieris rapae L. PPO from the fifth instar of Pieris rapae L. was purified using ammonium sulfate fractionation and chromatography on Sephadex G-100. The enzyme kinetics was characterized using L-3,4-dihydroxyphenylalanine （L-DOPA） as substrate. The results show that benzaldehyde, phydroxybenzaldehyde, p-chlorobenzaldehyde, and p-cyanobenzaldehyde can inhibit the PPO activity for the oxidation of L-DOPA. The inhibitor concentration leading to 50% activity lost, IC50, was estimated to be 5.90, 5.62, 2.83, and 2.91 mmol/L for the four tested inhibitors, respectively. Kinetic analyses show that the inhibitory effects of these compounds are reversible. Benzaldehyde, phydroxybenzaldehyde, and p-chlorobenzaldehyde are noncompetitive inhibitors while p-cyanobenzaldehyde is a mixed-type inhibitor. The inhibition constants were determined for all four inhibitors. p-chlorobenzaldehyde and p-cyanobenzaldehyde were more potent inhibitors than the other compounds. These results provide a basis for developing PPO inhibition-based pesticides.

Polyphenolic Compound and the Degree of Browning in Processing Apple Varieties

Polyphenolic compound in processing apple （Malus domestica Borkh.） varieties and the relationship between polyphenol content and enzymatic browning were studied to provide reference for raw material selection and processing method optimization. The content of polyphenol compound in 10 processing apple varieties （4 cider and 6 juice varieties） were analyzed using the Folin-Ciocalteu method and HPLC. The degree of browning and the activities of polyphenol oxidase were also studied. The content and proportion of the polyphenol varied depending on the variety. Bitter varieties globally showed a higher polyphenol concentration than sweet or acid varieties. Proanthocyanidins, chlorogenic acid, （＋）-catechin, （-）-epicatechin were high-concentrated polyphenols in apple fruits. Phloridzin, the unique polyphenol of apple, was abundant in the bitter variety Frequin rouge fruit. Total polyphenols, proanthocyanidins, （＋）-catechin, and phloridzin had higher correlations with browning. The correlation was low between chlorogenic acid and browning. The polyphenolic profiles were correlated with the apple types. Cider apples contained more polyphenol than juice apple varieties. The content of flavan-3-ol has a close relationship with fruit browning.

Agrobacterium tumefaciens-mediated transformation of embryogenic callus and CRISPR/Cas9-mediated genome editing in‘Feizixiao'litchi

Litchi(Litchi chinensis Sonn.)is a type of commercially prevalent subtropical and tropical fruit.Since litchi has a highly heterozygous genetic background and a long reproductive cycle,conventional breeding methods(such as hybridization)have limited ability to nurture new litchi cultivars.Here,an efficient and stable Agrobacterium tumefaciens-mediated genetic transformation of embryogenic callus was established in‘Feizixiao’litchi.Transgenic materials were verified using polymerase chain reaction(PCR)analysis,β-glucuronidase(GUS)assay,and green fluorescent protein(GFP)assay.To implement the technology of the Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)/associated protein 9(CRISPR/Cas9)technology in‘Feizixiao’litchi and verify the validity of these transformation systems,the litchi polyphenol oxidase gene(LcPPO,JF926153)was knocked out.Various categories of mutations,covering base insertions,deletions,and substitutions,were found in transgenic materials via sequence analysis.The transformation system achieved high feasibility and efficiency,and the system of CRISPR/Cas9 was successfully employed to edit genes in‘Feizixiao’litchi.This work provides an essential foundation for investigating the functions of genes and accelerating litchi genetic improvement.

Widely Untargeted Metabolome Profiling Provides Insight into Browning and Nutritional Quality Changes in Short-Term Stored Fresh-Cut Potato(Solanum tuberosum L.)Shreds

Potato(Solanum tuberosum L.)is susceptible to enzymatic browning after fresh processing,resulting in color change and potential alteration in the nutritional quality.In this study,a popular potato cultivar,Feiwuruita,was used to profile the metabolites involved in color and nutritional quality changes in fresh shreds stored at 0 and 4 h at 25°C(designated CK and CK4H,respectively).The shreds turned brown within 4 h of storage.In all,723 metabolites consisting 12 classes of compounds were detected in the samples,largely lipids,phenolic acids,alkaloids,amino acids and derivatives,flavonoids,organic acids,nucleotides and derivatives.Of these,163 metabolites accumulated differentially between CK and CK4H shreds.Polyphenolic compounds(phenolic acids and flavonoids)mostly increased in the shreds after 4 h storage.Conversely,the short-term storage drastically reduced lipid compounds(25 LysoPC and 19 LysoPE),while essential alkaloids and terpenoid compounds that are beneficial to human health increased in accumulation.The findings present global metabolome and nutritional composition changes in short-term stored shreds of Feiwuruita.This study provides important foundation for future studies on browning prevention/reduction and for better utilization of Feiwuruita.

Response dynamics of three defense related enzymes in cotton leaves to the interactive stress of Helicoverpa armigera (Hübner) herbivory and omethoate application

In order to explore the response dynamics of the activities of defense related enzymes in cotton leaves towards the interactive stress of Helicoverpa armigera herbivory and omethoate application, the activities of phenylalanine ammonia-lyase（PAL）, lipoxygenase（LOX）, and polyphenol oxidase（PPO） were examined from 6 to 126 h after cotton leaves were treated 12 h of H. armigera herbivory, and then sprayed with 800 mg L–1 omethoate. The results showed that the changes in the activities of PAL, LOX and PPO that occured under the interactive stress of H. armigera herbivory and omethoate application reflected the interactive effects of the two stresses on cotton defense. The similarity between the response dynamics of PAL, LOX, and PPO activities in cotton leaves under the interactive stress and that under H. armigera herbivory treatment alone showed that the induction of H. armigera herbivory on the activities of PAL, LOX and PPO in cotton leaves played a leading role in the interactive effects, and the effect of omethoate application played only a minor role. A joint factor analysis was performed according to a method which has been used to analyze the joint toxicity of pesticides; this analysis sought to clarify if there was a synergistic, antagonistic, or additive effect on PAL, LOX, and PPO activity in cotton leaves resulting from the interactive H. armigera herbivory and omethoate treatment. In the interactive effect on the response of PAL activity in cotton leaves, antagonistic effects of the omethoate application towards H. armigera herbivory were observed at 6 and 12 h. Synergistic effects were then observed at 18 and 30 h. Antagonistic effects were observed from 54 to 78 h and synergistic effects were finally observed at 126 h. The correlation between H. armigera herbivory and omethoate application in the interactive effect on cotton defense responses of LOX activity also fluctuated from synergism to antagonism during the time course. In the interactive effect on PPO activity, only antagonism was observed between H. armigera herbivory and omethoate application. In the interactive stress of H. armigera herbivory and omethoate application on cotton defense responses, omethoate affected the defense responses of cotton to H. armigera herbivory by producing antagonistic and synergistic effects. These results will be useful to understand the relationship between host plant and herbivorous pest.

Ethanol shock enhances the recovery of anthocyanin from lowbush blueberry

Deactivation of polyphenol oxidase(PPO)in natural products is essential for downstream processing of functional molecules used as food or food additives,particularly those served as antioxidants.In the present work,we identified two proteins with PPO activity from lowbush blueberry using ammonium sulphate precipitation and chromatography procedures.Deactivation of these proteins was studied using aqueous solutions of ethanol of different concentrations.The PPO activity was recovered after ethanol removal for the protein samples previously soaked in a lowconcentration ethanol solution.A complete and unrecoverable deactivation of the proteins was achieved using ethanol with concentration over 70%(v/v),as manifested by the significant changes in circular dichroism(CD)and fluorescence spectroscopy measurements.Based on these findings,we propose a new extraction process for blueberry anthocyanin,in which an ethanol shock,i.e.soaking blueberry fruit in a 70%(v/v)ethanol solution for 1 h,is implemented before subsequent procedures.This new process increases the anthocyanin yield by 55%in comparison to that without the ethanol shock.

Induction of Biochemical Changes in Santa Teriza Lime Leaves by Chitosan Application Influence Citrus Leaf Miner Damage

The effect of foliar spray of young lemon trees with Chitosan (CH) (200 - 300 - 400 ppm), on antioxidant enzymes activities (peroxidase POD, polyphenol oxidase PPO) and total phenolic content, and their influence on leaf miner (Phyllocnistis citrella) activity (tunnel length and damage density) under field condition was investigated. The results showed that treatment with chitosan 300 ppm enhanced the total phenolic content (5.975 mg/g) and the POD activity (0.533 min-1 mg-1 protein), while PPO activity was increased by chitosan 200 ppm (1.394 ΔA/min/g FW). The results demonstrated as well that chitosan treatment has a beneficial effect in reducing leaf miner activity, by decreasing tunnel length and damage density.

Enzyme from Banana(Musa sp.)Extraction Procedures for Sensitive Adrenaline Biosensor Construction

For building a biosensor, at least two enzyme sources can be employed. The pure enzyme has features with better selectivity and low stability. Crude extract presents better stability and wrong selectivity. Thus, one intermediate condition can be feasible joining both benefits of crude extract and pure enzymes. For that result, several procedures of extraction and semi purification of polyphenol oxidase (PPO) enzyme from banana (Musa sp.) were studied. The results showed that cleaned enzymatic extracts presented higher specific activities than crude extracts (2.8 up to 5.3 fold), despite the total protein concentration diminishing from 27% up to 72%, indicating that polyphenol oxidase (PPO) enzyme was preserved in them. The biosensors with 125 AU mL-1 containing cleaned enzymatic extracts performed better by grinding or grinding plus sonication for 30 s. They were linear over the ranges of 5.9 × 10-6 mol·L-1 to 1.4 × 10-3 mol·L-1 and 7.9 × 10-8 mol·L-1 to 4.0 × 10-3 mol·L-1, respectively. The limit of Detection (LOD) was 5.9 × 10-6 mol·L-1 and 7.9 × 10-8 mol·L-1, respectively. The LOD obtained is adequate to adrenaline determination on blood and medicinal samples, and were applied in medicinal samples with satisfactory results.

Lysozyme inhibits postharvest physiological deterioration of cassava

After harvest,cassava(Manihot esculenta Crantz)storage roots undergo rapid postharvest physiological deterioration,producing blue-brown discoloration in the vasculature due to the production of polyphenolics(mainly quinonesand coumarins)by enzymes such as polyphenol oxidase(PPO).Here,we report the application of hen egg-white lysozyme(HEWL),a natural PPO inhibitor,in transgenic cassava to repress the symptoms of postharvest physiological deterioration.The HEWL-expressing transgenic plants had lower levels of the two main cassava coumarins tested,scopoletin and scopolin,compared with wild type.HEWL-expressing cassava also showed increased tolerance of oxidative stress.Overall,the lysozyme-PPO system proved to be functional in plants for repressing PPO-mediated commercial product browning.