Partial purification of latent persimmon fruit polyphenol oxidase

Persimmon fruit polyphenol oxidase (PPO) was partially purified using a combination of phase partitioning with Triton X-114 and ammonium sulfate fractionation between 50 and 75%. The enzyme, which showed both monophenolase and diphenolase activities, was partially purified in a latent form and could be optimally activated by the presence of 1 mM sodium dodecyl sulfate (SDS) with an optimum pH of 5.5. In the absence of SDS, the enzyme showed maximum activity at acid pH. SDS-PAGE showed the presence of a single band when L-DOPA was used as substrate. The apparent kinetic parameters of the latent enzyme were determined at pH 5.5, the V(m) value being 15 times higher in the presence of SDS than in its absence, whereas the K(M) was the same in both cases, with a value of 0.68 mM. The effect of several inhibitors was studied, tropolone being the most active with a K(i) value of 0.45 microM. In addition, the effect of cyclodextrins (CDs) was studied, and the complexation constant (K(c)) between 4-tert-butylcatechol (TBC) and CDs was calculated using an enzymatic method. The value obtained for K(c) was 15580 M(-1).     

A review on spectrophotometric methods for measuring the monophenolase and diphenolase activities of tyrosinase

Tyrosinase is a copper enzyme with broad substrate specifity toward a lot of phenols with different biotechnological applications. The availability of quick and reliable measurement methods of the enzymatic activity of tyrosinase is of outstanding interest. A series of spectrophotometric methods for determining the monophenolase and diphenolase activities of tyrosinase are discussed. The product of both reactions is the o-quinone of the corresponding monophenol/diphenol. According to the stability and properties of the o-quinone, the substrate is classified as four substrate types. For each of these substrate types, we indicate the best method for measuring diphenolase activity (among eight methods) and, when applicable, for measuring monophenolase activity (among four methods). The analytical and numerical solutions to the system of differential equations corresponding to the reaction mechanism of each case confirm the underlying validity of the different spectrophotometric methods proposed for the kinetic characterization of tyrosinase in its action on different substrates.     

Cloning, expression, and characterization of a D-psicose 3-epimerase from Clostridium cellulolyticum H10

The noncharacterized protein ACL75304 encoded by the gene Ccel_0941 from Clostridium cellulolyticum H10 (ATCC 35319), previously proposed as the xylose isomerase domain protein TIM barrel, was cloned and expressed in Escherichia coli . The expressed enzyme was purified by nickel-affinity chromatography with electrophoretic homogeneity and then characterized as d-psicose 3-epimerase. The enzyme was strictly metal-dependent and showed a maximal activity in the presence of Co(2+). The optimum pH and temperature for enzyme activity were 55 °C and pH 8.0. The half-lives for the enzyme at 60 °C were 6.8 h and 10 min when incubated with and without Co(2+), respectively, suggesting that this enzyme was extremely thermostable in the presence of Co(2+) but readily inactivated without metal ion. The Michaelis-Menten constant (K(m)), turnover number (k(cat)), and catalytic efficiency (k(cat)/K(m)) values of the enzyme for substrate d-psicose were estimated to be 17.4 mM, 3243.4 min(-1), and 186.4 mM min(-1), respectively. The enzyme carried out the epimerization of d-fructose to d-psicose with a conversion yield of 32% under optimal conditions, suggesting that the enzyme is a potential d-psicose producer.     

Screening of postharvest agricultural wastes as alternative sources of peroxidases: characterization and kinetics of a novel peroxidase from lentil ( Lens culinaris L.) stubble

Aqueous crude extracts of a series of plant wastes (agricultural, wild plants, residues from sports activities (grass), ornamental residues (gardens)) from 17 different plant species representative of the typical biodiversity of the Iberian peninsula were investigated as new sources of peroxidases (EC 1.11.1.7). Of these, lentil (Lens culinaris L.) stubble crude extract was seen to provide one of the highest specific peroxidase activities, catalyzing the oxidation of guaiacol in the presence of hydrogen peroxide to tetraguaiacol, and was used for further studies. For the optimum extraction conditions found, the peroxidase activity in this crude extract (110 U mL(-1)) did not vary for at least 15 months when stored at 4 °C (k(inact) = 0.146 year(-1), t(1/2 inact) = 4.75 year), whereas, for comparative purposes, the peroxidase activity (60 U mL(-1)) of horseradish (Armoracia rusticana L.) root crude extract, obtained and stored under the same conditions, showed much faster inactivation kinetics (k(inact) = 2.2 × 10(-3) day(-1), t(1/2 inact) = 315 days). Using guaiacol as an H donor and a universal buffer (see above), all crude extract samples exhibited the highest peroxidase activity in the pH range between 4 and 7. Once semipurified by passing the crude extract through hydrophobic chromatography on phenyl-Sepharose CL-4B, the novel peroxidase (LSP) was characterized as having a purity number (RZ) of 2.5 and three SDS-PAGE electrophoretic bands corresponding to molecular masses of 52, 35, and 18 kDa. The steady-state kinetic study carried out on the H(2)O(2)-mediated oxidation of guaiacol by the catalytic action of this partially purified peroxidase pointed to apparent Michaelian kinetic behavior (K(m)(appH(2)O(2)) = 1.87 mM; V(max)(appH(2)O(2)) = 6.4 mM min(-1); K(m)(app guaicol) = 32 mM; V(max)(app guaicol) = 9.1 mM min(-1)), compatible with the two-substrate ping-pong mechanism generally accepted for peroxidases. Finally, after the effectiveness of the crude extracts of LSP in oxidizing and removing from solution a series of last-generation dyes present in effluents from textile industries (1) had been checked, a steady-state kinetic study of the H(2)O(2)-mediated oxidation and decolorization of Green Domalan BL by the catalytic action of the lentil stubble extract was carried out, with the observation of the same apparent Michaelian kinetic behavior (K(m)(appGD) = 471 μM; V(max)(appGD)= 23 μM min(-1)). Further studies are currently under way to address the application of this LSP crude extract for the clinical and biochemical analysis of biomarkers.     

Properties of diphenolase from Vanilla planifolia (Andr.) shoot primordia cultured in vitro.

Properties of diphenolase (PPO, EC1.10.3.1) from vanilla (Vanilla planifolia Andr.) shoot primordia culture were investigated. Two pH optima of the enzyme extraction at pH 6 and 8 were found. Nevertheless, the enzymes shared the same optimum pH of activity-between pH 3 and 4. Sodium dodecyl sulfate slightly improved diphenolase extraction but caused a 3-fold increase in its specific activity. The extracts of pH 6 and 8.0 revealed three isozyme bands after polyacrylamide gel electrophoresis-two of them were similar in both extracts and two distinct. The enzyme showed high thermal stability-no loss was observed after 120 min at 50 degrees C. Diethyldithiocarbamic acid, ethylenediaminetetracetic acid disodium salt, ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid, L-ascorbic acid, dithiothreitol, glutathione (reduced), and beta-mercaptoethanol were found to be potent inhibitors of the diphenolase studied. The enzyme showed also monophenolase activity. Km and Vmax were calculated with monophenols [p-coumaric acid, 3-(p-hydroxyphenyl)propionic acid, 4-hydroxybenzyl alcohol, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid] and with diphenols (caffeic acid, hydrocaffeic acid, chlorogenic acid, 4-methylcatechol, protocatechuic aldehyde and acid, and 3,4-dihydroxyphenylalanine). The highest Vmax was found with 4-hydroxybenzyl alcohol and the greatest affinity to protocatechuic acid, respectively-the most abundant monophenol and one of the least abundant o-diphenols in the studied Vanilla tissue.     

鸟粪石沉淀法脱除氨氮对鸡粪厌氧发酵过程的影响

为缓解鸡粪厌氧发酵过程中产生的氨氮抑制,采用投加镁磷盐的方式,在厌氧发酵过程中原位脱除氨氮,考察鸟粪石沉淀法脱除氨氮对鸡粪厌氧发酵过程的影响及镁磷盐的利用效率。试验向稳定运行的半连续厌氧反应器内投加MgCl2·6H2O和K2HPO4·3H2O,理论脱除速率为3 000 mg/d。第一次加盐脱除氨氮后,试验组反应器内氨氮浓度由2 937 mg/L降低至1 466 mg/L,平均产甲烷量为0.39 L/g,相较对照组的0.33 L/g提高了18%,镁磷盐利用率为91%;第二次加盐脱除氨氮后,试验组氨氮浓度由2 232 mg/L降低至762 mg/L,平均产甲烷量为0.33 L/g,相较对照组的0.30 L/g提高了10%,镁磷盐利用率为90%。研究表明鸟粪石沉淀法能较好的与厌氧发酵过程相耦合,在脱除氨氮缓解抑制的同时,提高系统甲烷产量,并回收部分氮磷资源。     

基于深度学习与目标跟踪的苹果检测与视频计数方法

基于机器视觉技术自动检测苹果树上的果实并进行计数是实现果园产量测量和智慧果园生产管理的关键。该研究基于现代种植模式下的富士苹果视频,提出基于轻量级目标检测网络YOLOv4-tiny和卡尔曼滤波跟踪算法的苹果检测与视频计数方法。使用YOLOv4-tiny检测视频中的苹果,对检测到的果实采用卡尔曼滤波算法进行预测跟踪,基于欧氏距离和重叠度匹配改进匈牙利算法对跟踪目标进行最优匹配。分别对算法的检测性能、跟踪性能和计数效果进行试验,结果表明：YOLOv4-tiny模型的平均检测精度达到94.47%,在果园视频中的检测准确度达到96.15%;基于改进的计数算法分别达到69.14%和79.60%的多目标跟踪准确度和精度,较改进前算法分别提高了26.86和20.78个百分点;改进后算法的平均计数精度达到81.94%。该研究方法可有效帮助果农掌握园中苹果数量,为现代化苹果园的测产研究提供技术参考,为果园的智慧管理提供科学决策依据。     

Studies on water transport through the sweet cherry fruit surface. 11. FeCl3 decreases water permeability of polar pathways

The effect of FeCl3 (10 mM) on osmotic water uptake into detached sweet cherry fruit (Prunus avium L.) and on the (3)H2O permeability (P(d)) of excised exocarp segments (ES) or enzymatically isolated cuticular membranes (CM) was investigated. ES or CM were mounted in an infinite dose diffusion system, where diffusion is monitored from a dilute donor solution through an interfacing ES or CM into a receiver solution under quasi steady-state conditions. In the absence of FeCl3, (3)H2O diffusion through stomatous ES was linear over time, indicating that P(d) was constant. Adding FeCl3 to the donor decreased P(d) by about 60%. P(d) remained at a decreased level when replacing the FeCl3 donor again by deionized water. The decrease in P(d) was positively and linearly related to the stomatal density of the ES. There was no effect of FeCl3 on the P(d) of astomatous sweet cherry fruit ES or CM regardless of the presence of wax (epicuticular or cuticular). FeCl3 decreased P(d) when added to the donor (-63%) or receiver (-16%), but there was no effect when it was added to donor and receiver solutions simultaneously. The decrease in P(d) depended on the pH of the receiver and the presence of citrate buffer. There was no effect of FeCl3 with citrate buffer as a receiver regardless of pH (range 2.0-6.0). When using nonbuffered receiver solutions with pH adjusted to pH 2.0, 3.0, 4.5, or 6.0, FeCl3 markedly decreased (3)H2O diffusion at pH > or = 3 but had no effect at pH 2.0. FeCl3 increased the energy of activation (E(a)) for (3)H2O diffusion (range 15-45 degrees C) through stomatous ES but had no significant effect in astomatous CM. The increase in E(a) by FeCl3 was positively related to stomatal density. FeCl3 decreased the P(d) for 2-(1-naphthyl)[1-(14)C]acetic acid (NAA) and 2,4-dichloro[U-(14)C]phenoxyacetic acid (2,4-D) in stomatous ES. The magnitude of the effect depended on the degree of dissociation and was larger for the dissociated acids (pH 6.2) than for the nondissociated acids (pH 2.2). Incubating whole fruit in isotonic solutions of selected osmotica resulted in significant water uptake that was inversely related to the molecular weight of the osmotica and was consistently lower for fruit treated with FeCl3. The FeCl3 induced decrease in water fluxes was larger for osmotica having a low molecular weight than for those with a higher molecular weight. Our data indicate that FeCl3 decreased the permeability of the stomatous sweet cherry exocarp to water and other polar substances by pH-dependent formation of precipitates that decrease transport along polar pathways. Decreasing the permeability of polar pathways by a precipitation reaction is a useful target in developing strategies against rain-induced fruit cracking.     

Characterization of monophenolase activity of polyphenol oxidase from iceberg lettuce

Polyphenol oxidase (EC 1.14.18.1), a thylakoid membrane-bound enzyme, was isolated by sonication of osmotically shocked chloroplasts from iceberg lettuce (Lactuca sativa). The enzyme showed monophenolase activity when assayed on (p-hydroxyphenyl)propionic acid with 3-methyl-2-benzothiazolinone hydrazone in a reliable continuous spectrophotometric method, with high sensitivity, accuracy, and precision. The monophenolase activity showed a lag period before the steady-state rate (V(ss)) was reached. Both kinetic parameters, the lag period and the steady-state rate, depended on the pH, the enzyme and substrate concentrations, and the presence of catalytic amounts of o-diphenol. This activity shows inhibition by high substrate concentration. The experimental results correspond with the mechanism previously described for PPO from other sources. Kinetic constants K(m), V(max), and K(i) were determined.     

Purification and characterization of a stable cysteine protease ervatamin B, with two disulfide bridges, from the latex of Ervatamia coronaria

Latex of the medicinal plant Ervatamia coronaria was found to contain at least three cysteine proteases with high proteolytic activity, called ervatamins. One of these proteases, named ervatamin B, has been purified to homogeneity using ion-exchange chromatography and crystallization. The molecular mass of the enzyme was estimated to be 26 000 Da by SDS-PAGE and gel filtration. The extinction coefficient (epsilon(1%)(280 nm)) of the enzyme was 20.5 with 7 tryptophan and 10 tyrosine residues per molecule. The enzyme hydrolyzed denatured natural substrates such as casein, azoalbumin, and azocasein with a high specific activity. In addition, it showed amidolytic activity toward N-succinyl-alanine-alanine-alanine-p-nitroanilide with an apparent K(m) and K(cat) of 6.6 +/- 0.5 mM and 1.87 x 10(2) s(-)(1), respectively. The pH optima was 6.0-6.5 with azocasein as substrate and 7.0-7.5 with azoalbumin as substrate. The temperature optimum was around 50-55 degrees C. The enzyme was basic with an isoelectric point of 9.35 and had no carbohydrate content. Both the proteolytic and amidolytic activity of the enzyme was strongly inhibited by thiol-specific inhibitors. Interestingly, the enzyme had only two disulfide bridges versus three as in most plant cysteine proteases of the papain superfamily. The enzyme was relatively stable toward pH, denaturants, temperature, and organic solvents. Polyclonal antibodies raised against the pure enzyme gave a single precipitin line in Ouchterlony's double immunodiffusion and typical color in ELISA. Other related proteases do not cross-react with the antisera to ervatamin B showing that the enzyme is immunologically distinct. The N-terminal sequence showed conserved amino acid residues and considerable similarity to typical plant cysteine proteases.     

pH and solubility of aluminium in acidic forest soils: a consequence of reactions between organic acidity and aluminium alkalinity

Data from two Podzol O and E horizons, sampled in 1-cm layers at 13 points within 2 m × 2 m plots, were used to test the hypothesis that the composition of hydrogen ions (H) and aluminium (Al) adsorbed to the solid-phase soil organic matter (SOM) determines pH and Al solubility in organic-rich acidic forest soils. Organically adsorbed Al was extracted sequentially with 0.5 m CuCl₂ and organically adsorbed H was determined as the difference between total acidity titrated to pH 8.2 and Al extracted in 0.5 m CuCl₂. The quotient between fractions of SOM sites binding Al and H (N_Al/N_H) is shown to determine the variation in pH and Al solubility. It is furthermore shown that models in which pH and Al solubility are linked via a pH-dependent solubility of an Al hydroxide and in which cation exchange between Al³⁺ and Ca²⁺, rather than cation exchange between Al³⁺ and H⁺, is the main pH-buffering process cannot be used to simulate pH or Al solubility in O and E horizons. The fraction of SOM sites adsorbing Al increased by depth in the lower O and throughout the E horizon at the same magnitude as sites adsorbing H decreased. The fraction of sites binding the cations Ca²⁺ + Mg²⁺ + K⁺ + Na⁺ remained constant. It is suggested that a net reaction between Al silicates (proton acceptors) and protonated functional groups in SOM (proton donors) is the long-term chemical process determining the composition of organically adsorbed H and Al in the lower part of the O and in the E horizon of Podzols. Thus, in the long term, pH and Al solubility are determined by the interaction between organic acidity and Al alkalinity.     

Leucine aminopeptidase from red sea bream (Pagrus major) skeletal muscle: purification, characterization, cellular location, and tissue distribution

A leucine aminopeptidase was purified for the first time from marine fish red sea bream ( Pagrus major) skeletal muscle to homogeneity with 4850-fold and a yield of 7.4%. The purification procedure consisted of ammonium sulfate fractionation and chromatographies including DEAE-Sephacel, Sephacryl S-200, hydroxyapatite, and phenyl-Sepharose. The enzyme was approximately 96 kDa as estimated by SDS-PAGE and gel filtration and preferentially hydrolyzed substrate Leu-MCA. The enzymatic activity was optimal at 45 degrees C and pH 7.5. The K m and k cat values of the enzyme for Leu-MCA were 1.55 microM and 26.4 S (-1) at 37 degrees C, respectively. Activation energy ( E a) of the enzyme was 59.6 kJ M (-1). The enzyme was specifically inhibited by metal-chelating agents, and Zn (2+) and (or) Mn (2+) seemed to be its metal cofactor(s). In addition, bestatin strongly inhibited its activity, and K i was 1.44 microM. Using a highly specific polyclonal antibody, the location of enzyme was demonstrated intracellularly and distributed in different tissues.     

Gene cloning, expression, and biochemical characterization of a recombinant trehalose synthase from Picrophilus torridus in Escherichia coli

A trehalose synthase (TSase) gene from a hyperacidophilic, thermophilic archaea, Picrophilus torridus, was synthesized using overlap extension PCR and transformed into Escherichia coli for expression. The purified recombinant P. torridus TSase (PTTS) showed an optimum pH and temperature of 6.0 and 45 degrees C, respectively, and the enzyme maintained high activity at pH 5.0 and 60 degrees C. Kinetic analysis showed that the enzyme has a 2.5-fold higher catalytic efficiency (k(cat)/K(M)) for maltose than for trehalose, indicating maltose as the preferred substrate. The maximum conversion rate of maltose into trehalose by the enzyme was independent of the substrate concentration, tended to increase at lower temperatures, and reached approximately 71% at 20 degrees C. Enzyme activity was inhibited by Hg2+, Al3+, and SDS. Five amino acid residues that are important for alpha-amylase family enzyme catalysis were shown to be conserved in PTTS (Asp203, Glu245, Asp311, His106, and His310) and required for its activity, suggesting this enzyme might employ a similar hydrolysis mechanism.     

Kinetic study of the quenching reaction of singlet oxygen by Pyrroloquinolinequinol (PQQH(2), a reduced form of Pyrroloquinolinequinone) in micellar solution

A kinetic study of the quenching reaction of singlet oxygen ((1)O(2)) with pyrroloquinolinequinol (PQQH(2), a reduced form of pyrroloquinolinequinone (PQQ)), PQQNa(2) (disodium salt of PQQ), and seven kinds of natural antioxidants (vitamin C (Vit C), uric acid (UA), epicatechin (EC), epigallocatechin (EGC), α-tocopherol (α-Toc), ubiquinol-10 (UQ(10)H(2)), and β-carotene (β-Car)) has been performed. The second-order rate constants k(Q) (k(Q) = k(q) + k(r), physical quenching and chemical reaction) for the reaction of (1)O(2) with PQQH(2), PQQNa(2), and seven kinds of antioxidants were measured in 5.0 wt % Triton X-100 micellar solution (pH 7.4), using UV-visible spectrophotometry. The k(Q) values decreased in the order of β-Car > PQQH(2) > α-Toc > UA > UQ(10)H(2) > Vit C ～ EGC > EC ? PQQNa(2). PQQH(2) is a water-soluble antioxidant. The singlet oxygen-quenching activity of PQQH(2) was found to be 6.3, 2.2, 6.1, and 22 times as large as the corresponding those of water-soluble antioxidants (Vit C, UA, EGC, and EC). Further, the activity of PQQH(2) was found to be 2.2 and 3.1 times as large as the corresponding activity of lipid-soluble antioxidants (α-Toc and UQ(10)H(2)). On the other hand, the activity of PQQH(2) is 6.4 times as small as that of β-Car. It was observed that the chemical reaction (k(r)) is almost negligible in the quenching reaction of (1)O(2) by PQQH(2). The result suggests that PQQH(2) may contribute to the protection of oxidative damage in biological systems, by quenching (1)O(2).     

Production, purification, and properties of an endoglucanase produced by the hyphomycete Chalara (Syn. Thielaviopsis) paradoxa CH32

The hyphomycete Chalara (syn. Thielaviopsis) paradoxa produces endoglucanase activity during the late trophophase. The low molecular mass (35 kDa) endoglucanase purified from cultured broths works optimally at 37 degrees C and pH 5.0. The enzyme inactivates at pH below 3.0 and also at temperatures of 50 degrees C or higher, but it is stable at lower temperatures, including refrigeration temperature and freezing. The enzyme is inhibited by detergents, by EDTA, and by the divalent cations Hg(2+) and Ag(2+). It is also inhibited to some extent by 10 mM Zn(2+), Fe(2+), and Mg(2+), but it is stimulated by Mn(2+). Enzyme activity is not affected by reducing agents. In the presence of low concentrations of water miscible organic solvents (20%) endoglucanase activity is inhibited by 7% (for methanol) to 50% (for acetonitrile), and it is totally inhibited at higher solvent concentrations (50%). Enzyme activity is not affected by the water immiscible solvent ethyl acetate. Carboxymethylcellulose is the preferred substrate (K(m(app)) = 8.3 g/L; V(max(app)) = 1.1 microM/min). Hydrolysis of crystalline cellulosic substrates is very limited, but it is greatly enhanced by phosphoric acid swelling. The purified enzyme shows no activity toward disaccharides or aryl-glucosides. Its activity is inhibited by cellobiose.     

中性电解水对鸡蛋表面的清洗灭菌效果

为寻求一种高效、安全、无污染的禽蛋清洗消毒剂,采用无隔膜电解装置电解稀盐酸溶液制备中性电解水（pH值6.0～7.5）考查不同有效氯浓度、处理时间和温度条件下中性电解水对鸡蛋人工接种鸡白痢沙门氏菌（Salmonella pullorum,鸡蛋表面的初始菌落数对数为6.19～6.26 log10 (cfu/g)）和大肠杆菌O157:H7（鸡蛋表面的初始菌落数对数为6.12～6.19 log10 (cfu/g)）的杀灭效果。结果表明,中性电解水对2种病菌均具有较强的杀灭效果,其杀菌效果随着有效氯浓度和处理时间的增加而增强,但温度对中性电解水的杀菌效果影响不显著。对菌悬液的杀菌试验表明：当中性电解水有效氯质量浓度为1.5 mg/L时,可以在20℃下3 min内完全杀灭鸡白痢沙门氏菌（初始含菌数的对数为 8.12 log10 (cfu/mL)）;质量浓度为2 mg/L时,可以100%杀灭大肠杆菌O157:H7（初始含菌数的对数为7.78 log10 (cfu/mL)）。当中性电解水清洗消毒被人工污染的鸡蛋表面时,有效氯质量浓度为12 mg/L、处理3 min可将鸡蛋表面的鸡白痢沙门氏菌全部杀灭,大肠杆菌O157:H7菌落数对数降低到1.0 log10 (cfu/g) 以下,且处理废液中没有残存菌,无二次污染问题。因此,中性电解水可以代替化学杀菌剂应用于鸡蛋清洗消毒。     

Purification and characterization of polyphenol oxidase from rape flower

The purification and partial enzymology characteristics of polyphenol oxidase (PPO) from rape flower were studied. After preliminary treatments, the crude enzyme solution was in turn purified with ammonium sulfate, dialysis, and Sephadex G-75 gel chromatography. The optimal conditions and stability of PPO were examined at different pH values and temperatures. Subsequently, PPO was also characterized by substrate (catechol) concentrations, inhibitors, kinetic parameters, and molecular weight. Results showed that the optimal pH for PPO activity was 5.5 in the presence of catechol and that PPO was relatively stable at pH 3.5-5.5. PPO was moderately stable at temperatures from 60 to 70 °C, whereas it was easily denatured at 80-90 °C. Ethylenediaminetetraacetic acid, sodium chloride, and calcium chloride had little inhibitive effects on PPO, whereas citric acid, sodium sulfite, and ascorbic acid had strongly inhibitive effects. The Michaelis-Menten constant (K(m)) and maximal reaction velocity (V(max)) of PPO were 0.767 mol/L and 0.519 Ab/min/mL of the crude PPO solution, respectively. PPO was finally purified to homogeneity with a purification factor of 4.41-fold and a recovery of 12.41%. Its molecular weight was 60.4 kDa, indicating that the PPO is a dimer. The data obtained in this research may help to prevent the enzymatic browning of rape flower during its storage and processing.     

Oxidation of salsolinol by banana pulp polyphenol oxidase and its kinetic synergism with dopamine

Salsolinol, a tethrahydroisoquinoline present in banana and biosynthesized from dopamine, was oxidized by banana pulp polyphenol oxidase to its corresponding salsolinol-o-quinone. This oxidation was pH-dependent and showed a maximum at acidic pH values. At physiological pH of 5.0, the values obtained for the kinetic parameter (V(m) and K(m)) were 62.5 microM/min and 1.7 mM, respectively. When dopamine was added to the reaction medium to imitate physiological conditions, salsolinol was co-oxidized by dopamine-quinone. When this phenomenon was studied oxygraphically, an unexpected activation of dopamine oxidation was found in the presence of salsolinol. This activation was related with the enzyme's kinetic mechanism and was named "kinetic synergism", because a bad substrate activated a good one. A possible physiological role is discussed.     

Effects of silicon on the germination,growth and physiology activity of Zea mays L. seedlings under PH stress

Silicon(Si) has a significant function in reducing abiotic stresses on plants. pH stress is one of abiotic stresses. We investigated the effects of silicon on maize seedlings under pH stress. The results showed that incorporation of Si (2.0?mM (mmol)) into pH 3.0 increased the growth, chlorophyll and carotenoid contents, decreased catalase, peroxidase and ascorbate peroxidase enzyme activity and malondialdehyde content. The combined treatments with Si (8.0?mM) and pH 3.0 decreased the maize growth compare with the single pH (3.0). Incorporation of Si (2.0 or 8.0?mM) into pH 8.0 were obviously unchanged compare with the single pH (8.0). The combined or single effects of Si (2.0 or 8.0?mM) and pH (3.0 or 8.0) on germination percentage were negligible. The application of Si (2.0?mM) could be a better strategy for improving the plant growth and alleviating low pH stress in soil.     

Modes of antifungal action of (2E)-alkenals against Saccharomyces cerevisiae

A series of aliphatic (2E)-alkenals from C(5) to C(14) were tested for their antifungal activity against Saccharomyces cerevisiae ATCC 7754. (2E)-Undecenal (C(11)) was found to be the most effective with the minimum fungicidal concentration (MFC) of 6.25 microgram/mL, followed by (2E)-decenal (C(10)) with an MFC of 12.5 microgram/mL. The time-kill curve study showed that (2E)-undecenal was fungicidal against S. cerevisiae at any growth stage, and this activity was not influenced by pH values. The (2E)-alkenals inhibited glucose-induced acidification by inhibiting the plasma membrane H(+)-ATPase. The primary antifungal action of medium-chain (C(9)-C(12)) (2E)-alkenals against S. cerevisiae comes from their ability to function as nonionic surface-active agents (surfactants), disrupting the native membrane-associated function nonspecifically. Hence, the antifungal activity of (2E)-alkenals is mediated by biophysical processes, and the maximum activity can be obtained when the balance between the hydrophilic and hydrophobic portions becomes the most appropriate.