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.

Microwave flow and conventional heating effects on the physicochemical properties, bioactive compounds and enzymatic activity of tomato puree.

BACKGROUND: Thermal processing causes a number of undesirable changes in physicochemical and bioactive properties of tomato products. Microwave (MW) technology is an emergent thermal industrial process that offers a rapid and uniform heating, high energy efficiency and high overall quality of the final product. The main quality changes of tomato puree after pasteurization at 96 ± 2 °C for 35 s, provided by a semi-industrial continuous microwave oven (MWP) under different doses (low power/long time to high power/short time) or by conventional method (CP) were studied. RESULTS: All heat treatments reduced colour quality, total antioxidant capacity and vitamin C, with a greater reduction in CP than in MWP. On the other hand, use of an MWP, in particular high power/short time (1900 W/180 s, 2700 W/160 s and 3150 W/150 s) enhanced the viscosity and lycopene extraction and decreased the enzyme residual activity better than with CP samples. For tomato puree, polygalacturonase was the more thermo-resistant enzyme, and could be used as an indicator of pasteurization efficiency. CONCLUSION: MWP was an excellent pasteurization technique that provided tomato puree with improved nutritional quality, reducing process times compared to the standard pasteurization process. © 2016 Society of Chemical Industry.

Flash photolysis of cutinase: identification and decay kinetics of transient intermediates formed upon UV excitation of aromatic residues.

Aromatic amino acids play an important role in ultraviolet (UV)-induced photochemical reactions in proteins. In this work, we aim at gaining insight into the photochemical reactions induced by near-UV light excitation of aromatic residues that lead to breakage of disulfide bridges in our model enzyme, Fusarium solani pisi cutinase, a lipolytic enzyme. With this purpose, we acquired transient absorption data of cutinase, with supplemental experimental data on tryptophan (Trp) and lysozyme as reference molecules. We here report formation kinetics and lifetimes of transient chemical species created upon UV excitation of aromatic residues in proteins. Two proteins, lysozyme and cutinase, as well as the free amino acid Trp, were studied under acidic, neutral, and alkaline conditions. The shortest-lived species is assigned to solvated electrons (lifetimes of a few microseconds to nanoseconds), whereas the longer-lived species are assigned to aromatic neutral and ionic radicals, Trp triplet states, and radical ionic disulphide bridges. The pH-dependent lifetimes of each species are reported. Solvated electrons ejected from the side chain of free Trp residues and aromatic residues in proteins were observed 12 ns after excitation, reaching a maximum yield after approximately 40 ns. It is interesting to note that the formation kinetics of solvated electrons is not pH-dependent and is similar in the different samples. On the other hand, a clear increase of the solvated electron lifetime is observed with increasing pH. This observation is correlated with H3O+ being an electron scavenger. Prolonged UV illumination of cutinase leads to a larger concentration of solvated electrons and to greater absorption at 410 nm (assigned to disulphide electron adduct RSSR *-), with concomitant faster decay kinetics and near disappearance of the Trp* radical peak at 330 nm, indicating possible additional formation of TyrO* formed upon reaction of Trp* with Tyr residues. Prolonged UV illumination of cutinase also leads to a larger concentration of free thiol groups, known to originate from the dissociation of RSSR *-. Additional mechanisms that may lead to the near disappearance of Trp(*) are discussed. Our study provides insight into one key UV-light-induced reaction in cutinase, i.e., light-induced disruption of disulphide bridges mediated by the excitation of aromatic residues. Knowledge about the nature of the formed species and their lifetimes is important for the understanding of UV-induced reactions in humans that lead to light-induced diseases, e.g., skin cancer and cataract formation.

Fluorescence of the single tryptophan of cutinase: temperature and pH effect on protein conformation and dynamics.

The cutinase from Fusarium solani pisi is an enzyme with a single L-tryptophan (Trp) involved in a hydrogen bond with an alanine (Ala) residue and located close to a cystine formed by a disulfide bridge between two cysteine (Cys) residues. The Cys strongly quenches the fluorescence of Trp by both static and dynamic quenching mechanisms. The Trp fluorescence intensity increases by about fourfold on protein melting because of the disruption of the Ala-Trp hydrogen bond that releases the Trp from the vicinity of the cystine residue. The Trp forms charge-transfer complexes with the disulfide bridge, which is disrupted by UV light irradiation of the protein. This results in a 10-fold increase of the Trp fluorescence quantum yield because of the suppression of the static quenching by the cystine residue. The Trp fluorescence anisotropy decays are similar to those in other proteins and were interpreted in terms of the wobbling-in-cone model. The long relaxation time is attributed to the Brownian rotational correlation time of the protein as a whole below the protein-melting temperature and to protein-backbone dynamics above it. The short relaxation time is related to the local motion of the Trp, whose mobility increases on protein denaturation.

Tryptophan mediated photoreduction of disulfide bond causes unusual fluorescence behaviour of Fusarium solani pisi cutinase.

The fluorescence signal of the single tryptophan residue (Trp69) of Fusarium solani pisi cutinase is highly quenched. However, prolonged irradiation of the enzyme in the tryptophan absorption band causes an increase of the tryptophan fluorescence quantum yield by an order of magnitude. By using a combination of NMR spectroscopy and chemical detection of free thiol groups with a sulfhydryl reagent we could unambiguously show that the unusual fluorescence behaviour of Trp69 in cutinase is caused by the breaking of the disulfide bond between Cys31 and Cys109 upon irradiation, while the amide-aromatic hydrogen bond between Ala32 and Trp69 remains intact. This is the first example of tryptophan mediated photoreduction of a disulfide bond in proteins.

Analysis of microsomal cholesteryl ester hydrolases by radiation inactivation.

Radiation inactivation by high energy electrons, a method for determining the size of a protein without prior purification, was used to study the acid and neutral cholesteryl ester hydrolase (CEH) activities of rat liver microsomes. The same preparations were also assayed for the microsomal, "nonspecific" carboxylesterases using o-nitrophenyl acetate as substrate. Non-specific esterase activity surviving radiation could be fit to a single exponential function, the slope of which yielded a target size of 47 +/- 5 kDa (mean +/- S.D., n = 7). Surviving CEH activity assayed at pH 5 could also be fit to a single exponential that yielded a target size of 71 +/- 14 kDa (n = 5). In contrast, the surviving CEH activity assayed at pH 7 was more complex. The data from six experiments were described as the sum of two exponentials, indicating that most of the activity is due to an entity that is three to four times larger and a minor amount to one that is half the size of the pH 5 enzyme. The results are consistent with the suggestion that the acid and neutral microsomal CEH activities are due to distinct enzymes, which are not the "nonspecific" carboxylesterases. Their sizes also differ from those previously determined for lysosomal acid lipase and other lipases in the liver.

Target size of neurotoxic esterase and acetylcholinesterase as determined by radiation inactivation.

The target size of neurotoxic esterase (NTE), the putative target site for the initiation of organophosphorus-compound-induced delayed neurotoxicity, and acetylcholinesterase (AChE) from hen brain were examined by determining the rate at which the activities of the esterases were destroyed by ionizing irradiation. Samples of hen brain were prepared by slowly drying a microsomal preparation under vacuum. The dried samples were then irradiated with electrons from a 1 MeV Van de Graaff generator. The doses ranged from 0 to 28 Mrad. The radiation doses were calibrated by the rate of inactivation of T1-bacteriophage plaque induction. Following the irradiation procedure, the samples were resuspended in buffer and enzymic activity was measured. The target size of NTE from hen brain was determined to be about 105 kDa, whereas hen brain AChE was found to have a target size of about 53 kDa. The target size of NTE was found to be similar in experiments with rat brain and cat brain. In addition, commercial preparations of electric-eel electric-organ AChE and horse serum butyrylcholinesterase were found to have target sizes that were identical with each other, and also were very similar to that of AChE from hen brain.

Changes of fluoresceindiacetate-hydrolysis and transport in mammalian cells after ultra-violet- and gamma irradiation.

Changes in the permeability of cell membranes and in the enzymatic activity of intact cells and their homogenates after irradiation were studied. CHO-cells, erythrocytes and their homogenates were irradiated with cobalt-gamma radiation and UV-light. It was found that the esterases are more radio-sensitive in intact cells than in the corresponding homogenates. The decrease of the enzymatic activity after irradiation can be described by a one-hit curve, whereas the loss of transport activity can be resolved into two one-hit curves. The slope associated with the more sensitive target mainly determines the course of the composed transport curve. Mechanisms for the decreases in the rate constants are discussed.