Effect of infrared radiation on activity and conformation of polyphenol oxidase from Acetes chinensis.

The aim of this paper was to study the effect of infrared radiation (IR) on the activity and conformation of polyphenol oxidase (PPO) in Acetes chinensis. In this paper, the specific activity of PPO was increased from 21.2 to 643.4 U/mg by a four-step purification. The results showed that IR treatment had greater effect on the enzyme activity and conformation of PPO than hot air (HA) treatment. After IR treatment at 70°C, the relative enzyme activity of PPO was 9.28%, the surface hydrophobicity index increased by 80.42%, and the content of sulfhydryl group decreased to 96.99% of the control group. The results of circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR) showed that the α-helix of PPO treated by IR decreased and the random coil increased. The intrinsic fluorescence intensity of PPO decreased after IR treatment, indicating that the tertiary structure of PPO was destroyed. Scanning electron microscopy (SEM) results showed that the surface microstructure of PPO after IR treatment became clear and compact. In conclusion, IR treatment can completely destroy the secondary structure and tertiary structure of PPO and cause enzyme inactivation. This study provides a treatment for reducing the activity of PPO from A. chinensis during the production and processing. PRACTICAL APPLICATION: This study shows that IR treatment has a better inhibitory effect on the activity of PPO than HA treatment. It provides a better treatment method for inactivating the activity of PPO from Acetes chinensis during the production and processing.

Effect of high pressure and thermal processing on spoilage-causing enzymes in mango (Mangifera indica).

The aim of the present work was to model the effect of combined pressure-temperature processing on spoilage-causing enzymes in mango pulp; which conventionally are inactivated using high temperatures leading to inevitable quality losses. The inactivation of enzymes pectin methylesterase (PME), polyphenol oxidase (PPO) and peroxidase (POD) was studied in mango pulp within the pressure, temperature and hold-time ranges of 0.1 to 600MPa, 40 to 70°C and 1s to 90min, respectively. The enzyme inactivation was described as a dual process: initial change in activity during dynamic pressure build-up phase and subsequent decrease under isobaric-isothermal conditions. The former led to considerable increase in activities of all the three enzymes (p<0.05); however, the increased activity reduced with increased intensity of applied pressure-temperature. On the other hand, isobaric-isothermal conditions led to substantial inactivation (p<0.05), with 600MPa/70°C/20min treatment being most effective in reducing the activities of PME, PPO and POD to 32, 15 and 26%, respectively. The enzyme inactivation data was non-linear under isobaric-isothermal conditions and fitted to the nth-order reaction model, indicative of the occurrence of series of reactions possibly due to pressure-temperature interaction effects. The estimated reaction order 'n' was 0.815, 1.106 and 1.137 for PME, PPO and POD, respectively. The estimated reaction rate constant k (min-1) depicted PME to be the most baroresistant enzyme followed by POD and PPO. Temperature and pressure dependency of k was expressed in terms of activation energy and activation volume using the Arrhenius- and Eyring-type relations, respectively. An empirical model with good correlation between actual and predicted data (R2>0.90) was proposed to simulate the rate of enzyme inactivation under isobaric-isothermal conditions as a function of pressure and temperature.

Inactivation of polyphenoloxidase by pulsed light.

The effect of pulsed light on the inactivation of polyphenoloxidase (PPO) in model solutions was investigated focusing on the effect of enzyme concentration and total energy dose of the treatment. PPO inactivation increased with the dose of the treatment. Complete enzyme inactivation was achieved by pulsed light doses higher than 8.75 J cm(-2) . At low PPO concentrations (4 to 10 U), the enzyme resulted highly inactivated by pulsed light treatment. Further increase in enzyme units determined a progressive decrease in PPO inactivation. The latter was attributed to protein structural modifications including cleavage and unfolding/aggregation phenomena. PPO amounts higher than 10 U probably favoured enzyme conformations that were less prone to intermolecular rearrangements leading to inactivation.

In situ inactivation of polyphenol oxidase in mamey fruit (Pouteria sapota) by microwave treatment.

Polyphenol oxidase (PPO) is the enzyme responsible for quality loss in most fruits and vegetables. Quality loss is mainly because of oxidative chemical reactions which generate the darkening of tissues. Mamey fruit (Pouteria sapota) after harvesting suffers a rapid quality decay trough activation of PPO. However, PPO may be inactivated in situ by chemical or thermal treatment. In food processing, microwave treatment (MT) has been used recently as an alternative for PPO inactivation. In this study, it was observed that mamey fruit pulp subjected to a gently MT resulted in a higher PPO activity as the generated heat induced in situ the increase in PPO activity. In contrast, PPO was completely inactivated after long MT by using a high microwave power. Temperature in mamey pulp after MT reached a maximum of 79 °C; although PPO was active up to 60 °C. PPO was completely inactivated when conventional blanching treatment was performed but required a higher temperature (92 °C/300 s). The optimum energy intensity (E(opt)) for PPO inactivation by MT was 0.51 kJ/g or 937 W/165 s. Under this condition, the remaining PPO activity was inversely proportional to energy intensity (E). Interestingly, MT resulted in a negligible damage in microstructure of mamey pulp, although blanching treatment resulted in large damaging effects on tissue organization and shape. Therefore, MT is proposed as an effective way to completely inactivate PPO without causing any significant damage to fruit tissues and shape; as preservation of color, flavor, and taste would be favored.

Cell phone radiations affect early growth of Vigna radiata (mung bean) through biochemical alterations.

The indiscriminate use of wireless technologies, particularly of cell phones, has increased the health risks among living organisms including plants. We investigated the impact of cell phone electromagentic field (EMF) radiations (power density, 8.55 microW cm(-2)) on germination, early growth, proteins and carbohydrate contents, and activities of some enzymes in Vigna radiata. Cell phone EMF radiations significantly reduced the seedling length and dry weight of V radiata after exposure for 0.5, 1, 2, and 4 h. Furthermore, the contents of proteins and carbohydrates were reduced in EMF-exposed plants. However, the activities of proteases, alpha-amylases, beta-amylases, polyphenol oxidases, and peroxidases were enhanced in EMF-exposed radicles indicating their role in providing protection against EMF-induced stress. The study concludes that cell phone EMFs impair early growth of V radiata seedlings by inducing biochemical changes.

Inactivation of Saccharomyces cerevisiae and polyphenoloxidase in mango nectar treated with UV light.

Fresh mango nectar was processed by UV light at five flow rates (0.073 to 0.451 liter/min) and five UV light doses (75 to 450 kJ/m2) to evaluate total microbial load, Saccharomyces cerevisiae survival, and polyphenoloxidase activity. UV systems containing an inner mercury lamp (254 nm) each with intensity of 25 mW/cm2 were used as germicidal sources. In addition, mango nectar was treated for 15 min at 0.073 and 0.451 liter/min, stored at 3 degrees C, and evaluated periodically for total microbial count, yeast count, color, and polyphenoloxidase activity. The first-order kinetics modeling found that DUV-values in mango nectar ranged from 27.9 to 10.9 min (R2 > 0.950) and 26.0 to 11.8 min (R2 > 0.962) for total microbial count and yeast count, respectively. The maximum log reduction (CFU per milliliter) was 2.71 and 2.94 for total microbial count and yeast count, respectively, after 30 min of UV treatment at 0.451 liter/min. DUV-values ranging from 156 to 204 min were observed for polyphenoloxidase activity. The remaining polyphenoloxidase activity after 30 min of UV treatment at 0.451 liter/min was 19 +/- 4%. Initial microbial load and yeast in stored mango nectar were reduced in the range 2.86 to 3.41 and 1.82 to 1.97 log (CFU/ml) cycles, respectively. No substantial microbial growth was observed prior to 20 days of storage. Averages of 1,055 +/- 32, 803 +/- 32, and 710 +/- 37 enzyme activity units were observed in mango nectar UV processed at 0, 0.073, and 0.451 liter/min, respectively, during the entire storage period. However, mango nectar treated at 0.073 and 0.451 liter/min maintained a yellow and yellow-orange color, respectively, after 26 days of storage.

Dose rate effect of gamma irradiation on phenolic compounds, polyphenol oxidase, and browning of mushrooms (Agaricus bisporus).

To enhance the shelf life of edible mature mushrooms, Agaricus bisporus, 2 kGy ionizing treatments were applied at two different dose rates: 4.5 kGy/h (I(-)) and 32 kGy/h (I(+)). Both I(+) and I(-) showed a 2 and 4 day shelf-life enhancement compared to the control (C). Before day 9, no significant difference (p>0.05) in L value was detected in irradiated mushrooms. However, after day 9, the highest observed L value (whiteness) was obtained for the mushrooms irradiated in I(-). Analyses of phenolic compounds revealed that mushrooms in I(-) contained more phenols than I(+) and C, the latter containing the lower level of phenols. The fluctuation of the precursors of glutaminyl-4-hydroxyaniline (GHB) was less in I(-) than in I(+). The polyphenol oxidase (PPO) activities of irradiated mushrooms, analyzed via catechol oxidase, dopa oxidase, and tyrosine hydroxylase substrates, were found to be significantly lowered (p = 0.05) compared to C, with a further decrease in I(+). Analyses of the enzymes indicated that PPO activity was lower in I(+), contrasting with its lower phenols concentration. The observation of mushrooms' cellular membranes, by electronic microscopy, revealed a better preserved integrity in I(-) than in I(+). It is thus assumed that the browning effect observed in I(+) was caused by both the decompartmentation of vacuolar phenol and the entry of molecular oxygen into the cell cytoplasm. The synergetic effect of the residual active PPO and the molecular oxygen, in contact with the phenols, allowed an increased oxidation rate and, therefore, a more pronounced browning I(+) than in I(-).

Thermal inactivation of mushroom polyphenoloxidase employing 2450 MHz microwave radiation.

Browning reactions in fruits and vegetables are a serious problem for the food industry. In mushrooms, the principal enzyme responsible for the browning reaction is polyphenoloxidase (PPO). A microwave applicator has been designed and used for studying mushroom PPO inactivation. The effects of microwaves and conventional heating on the kinetics of the monophenolase and diphenolase activities of PPO were studied. Conventional and microwave treatments produce different enzyme intermediates with different stability and kinetic properties. We describe how considerable time can be saved during microwave inactivation of the enzyme compared with the time needed when conventional hot-water treatment is used, resulting in greater profitability and enhanced quality. The short exposure time required for samples irradiated with microwaves is very important for maintaining the quality of mushrooms. The fast microwave treatment used resulted in an increase in antioxidant content and a considerable decrease in browning.

Radiation-damaged tyrosinase molecules are inactive.

Target analysis of radiation inactivation of mushroom tyrosinase yields different target sizes for diphenoloxidase and monophenoloxidase activities, which correspond to the subunits H and HL2 (or HL), respectively. After gel electrophoresis of irradiated samples, all diphenoloxidase activity is observed at the same position as seen in the original material. Radiolytic fragments contain no detectable activity, consistent with a fundamental assumption of target theory.

Photoinactivation of agaricus bisporus tyrosinase: modification of the binuclear copper site.

Irradiation of Agaricus bisporus tyrosinase in the presence of citrate at pH 5.6 with 300-420-nm light results in a loss of both catecholase activity and cresolase activity. The light-sensitive species appears to be an enzyme-citrate complex, most likely involving coordination of citrate to the active site copper. One copper ion from each binuclear active site can be removed from the inactivated enzyme, resulting in the formation of a met apo derivative. The electron spin resonance spectrum of met apo tyrosinase resembles that of met apo hemocyanin and half-met Neurospora tyrosinase. It is consistent with a distorted square-planar geometry around the copper and with either nitrogen or nitrogen and oxygen ligands. Amino acid analysis indicates that four histidines on the heavy subunit are destroyed during the inactivation process. Some or all of these histidines may serve as ligands to the copper ion which becomes labile after inactivation. Photoinactivation results in decarboxylation of citrate and does not require the presence of oxygen. The reaction may involve generation of a free radical from the citrate which then attacks nearby histidine residues.

Light-induced enzyme system leading to pigmentation.

Alpha-Chymotrypsin was light sensitized by acylating with cis-cinnamoyl ester, a substrate interconvertible to the trans form by ultraviolet (UV) light. The degree of acylation by this method was complete leaving no residual activity of the enzyme. Upon UV irradiation the inhibited enzyme regained about 70% of its original activity, thereby adding light-sensitiveness to the proteolytic enzyme. In seeking a photographic application of the light-sensitized enzyme, a pigmenting enzyme was incorporated with it. The coupled enzyme system was shown to exhibit a light signal in the form of dark pigment slurry.

Photoreduction of copper chromophores in blue oxidases.

The low temperature (77 K) irradiation of oxidized ceruloplasmin and Rhus vernicifera laccase at the 330 nm absorption which arises from type 3 copper leads to the reduction of type 1 copper as demonstrated by bleaching of the 610 nm chromophore and the decrease of the EPR signal associated with this species. Type 2 copper remains unaffected. Concomitant with the type 1 copper reduction, a new EPR signal which is possibly that of a biradical appears. Upon thawing, type 1 copper is reversibly oxidized and the radical signal disappears. Irradiation of oxidized protein at the absorption band of type 1 copper produces no spectral change. An EPR study at room temperature confirms the wave-length specificity and reversibility of the photoreduction of type 1 copper and radical formation. Radical appearance and disappearance at room temperature are extremely slow (tau1/2 approximately 30 min). Optical studies at room temperature show that upon anaerobic irradiation of laccase in the 330 nm absorption band, both type 3 and type 1 chromophores are slowly reduced. Upon return to the dark and in the presence of O2, both type 3 and type 1 centers are reoxidized. Oxidizing equivalents either from O2 or K3Fe(CN)6 are required for the reoxidation reaction. These studies demonstrate that there is a direct energy transfer between type 3 and type 1 copper sites in blue copper oxidases.