Polyphenol oxidase activity,phenolic acid composition and browning in cashew apple (Anacardium occidentale, L.) after processing

This study describes the extraction and characterisation of cashew apple polyphenol oxidase (PPO) and the effect of wounding on cashew apple phenolic acid composition, PPO activity and fruit browning. Purification factor was 59 at 95% (NH₄)₂SO₄ saturation. For PPO activity, the optimal substrate was catechol and the optimum pH was 6.5. PPO K_m and V_max values were 18.8 mM and 13.6 U min⁻¹ ml⁻¹, respectively. Ascorbic acid, citric acid, sodium sulphite and sodium metabisulphite decreased PPO activity, while sodium chloride increased PPO activity. Wounding at 2 °C and 27 °C for 24 h increased PPO activity but storage at 40 °C reduced PPO activity. Gallic acid, protocatechuic acid and cinnamic acid (free and conjugate) were identified in cashew apple juice. Cutting and subsequent storage at 40 °C hydrolysed cinnamic acid. 5-Hydroxymethylfurfural content in cashew apple juice increased after injury and storage at higher temperatures, indicating non-enzymatic browning.     

Quantification and characterisation of polyphenol oxidase from vanilla bean

Polyphenol oxidase (PPO) of Vanilla planifolia Andrews beans was extracted and purified through ammonium sulphate precipitation, dialysis, and gel filtration chromatography. PPO activity was measured by improved UV technique using 4-methylcatechol and catechol as substrates increasing substantial sensitivity of previous procedure. The optimum pH and temperature for PPO activity were found to be 3.0 and 3.4 and 37 °C, respectively. K_m and V_max values were found to be 10.6 mM/L and 13.9 OD₃₀₀ min⁻¹ for 4-methylcatechol and 85 mM/L and 107.2 OD₃₀₀ min⁻¹ for catechol. In an inhibition test, the most potent inhibitor was found to be 4-hexylresorcinol followed by ascorbic acid. The thermal inactivation curve was biphasic. Activation energy (E_a) and z values were calculated as 92.10 kJ mol⁻¹ and 21 °C, respectively.     

Characterization of polyphenol oxidase from butter lettuce (Lactuca sativa var. capitata L.)

Polyphenol oxidase (PPO) was isolated from butter lettuce (Lactuca sativa var. capitata L.) grown in Poland and its biochemical characteristic were studied. PPO from butter lettuce showed a higher affinity to 4-methylcatechol than to catechol. The K_M and V_max values were: 3.20 ± 0.01 mM and 4081 ± 8 U/ml min⁻¹ for catechol and 1.00 ± 0.09 mM and 5405 ± 3 U/ml min⁻¹ for 4-methylcatechol. The optimum pHs of the enzyme were found to be 5.5 using catechol and 6.8 using 4-methylcatechol as substrate. The enzyme had a temperature optimum of 35 °C. The enzyme was relatively stable at 30 °C and 40 °C. The times required for 50% inactivation of activity at 50 °C, 60 °C and 70 °C were found to be about 30, 20 and 5 min, respectively. Inhibitors used for investigation in this study were placed in relative order of inhibition: p-hydroxybenzoic acid > glutathione ≈ ascorbic acid > l-cysteine > EDTA > citric acid. The enzyme eluted in the chromatographic separations was analyzed electrophoretically under denaturating conditions. The analysis revealed a single band on the SDS–PAGE which corresponded to a molecular weight of 60 kDa.     

Kinetic characterisation and thermal inactivation study of polyphenol oxidase and peroxidase from table grape (Crimson Seedless)

Polyphenol oxidase (PPO) and peroxidase (POD) were extracted from a table grape (Crimson Seedless) using Triton X-114 and characterized using spectrophotometric methods. Both PPO and POD were activated by acid shock. However, in the presence of the anionic detergent sodium dodecil sulphate (SDS), PPO was activated whereas POD was inactivated. The enzymes were kinetically characterized and both followed Michaelis–Menten kinetics, although with different values of their kinetic parameters. The V_m/K_m ratio showed that Crimson Seedless grape PPO presents a similar affinity for 4-tert-butyl-catechol (TBC) whether activated by acid shock (0.018 min⁻¹) or SDS (0.023 min⁻¹). With regards to POD, the K_m and V_m values for 2,2′-azinobis(3-ethylbenzothiazolinesulphonic acid) (ABTS) were 0.79 mM and 1.20 μM/min, respectively. In the case of H₂O₂, the K_m and V_m value were 0.4 mM and 0.93 μM/min, respectively. PPO and POD showed similar thermostability, losing >90% of relative activity after only 5 min of incubation at 78 °C and 75 °C, respectively. In addition, PPO´s activation energy was similar to that obtained for POD (295.5 kJ/mol and 271.9 kJ/mol, respectively).     

Immobilization of pepsin on chitosan beads

In this study, chitosan beads were prepared by using a cross-linking agent and the resulting beads were employed in immobilization process. Studies on free and immobilized pepsin systems for determination of optimum temperature, optimum pH, thermal stability, pH stability, operational stability, storage stability and kinetic parameters were carried out. The optimum temperature interval for free pepsin and immobilized pepsin were 30–40 and 40–50 °C, respectively. Free and immobilized pepsin showed higher activity at pH 2.0–4.0. Apparent K_m = 12.0 g L⁻¹ haemoglobin (1.56 mM tyrosine) and V_max = 5220 μmol (mg protein min)⁻¹ values were obtained for free pepsin, while apparent K_m = 20.0 g L⁻¹ haemoglobin (2.16 mM tyrosine) and V_max = 2780 μmol (mg protein min)⁻¹ values were obtained for immobilized pepsin. Thermal stability and storage stability of immobilized pepsin were higher than that of free pepsin. Milk clotting activity was used for evaluation of the applicability of pepsin immobilization to industrial process. Optimum milk clotting temperature was found as 40 °C for free pepsin and 50 °C for immobilized pepsin.     

Purification and characterization of polyphenol oxidase from Ferula sp.

Polyphenol oxidase (PPO) of several Ferula sp. was extracted and purified through (NH₄)₂SO₄ precipitation, dialysis, and gel filtration chromatography. Leaf and stem extracts were used for the determination of enzyme properties. Optimum conditions, for pH, temperature, and ionic strength were determined. The best substrates of PPO were catechol for leaf and (−) epicatechin for stem samples. Optimum pH and temperature were determined. K_M and V_max values were 2.34 × 10⁻³ M and 8541 EU/ml for catechol, and 2.89 × 10⁻³ M and 5308 EU/ml for (−) epicatechin. The most effective inhibitor was sodium diethyl dithiocarbamate for leaf samples and sodium metabisulphite for stem samples. Both inhibitors indicated competitive reactions. PPO showed irreversible denaturation after 40 min at 60 °C.     

Extraction and characterisation of pectin methylesterase from black carrot (Daucus carota L.)

This study was carried out to determine some of the biochemical properties of pectin methylesterase (PME) from black carrot. The enzyme showed very high activity in a broad pH range of 6.5–8.5, with the optimum pH occurring at 7.5. The optimum temperature for maximal PME activity was found to be 55 °C. NaCl enhanced PME activity, particularly at 0.2 M. K_m and V_max values for black carrot PME using apple pectin as substrate were found to be 2.14 mg/ml (r² = 0.988) and 3.75 units/ml, respectively. The enzyme was stable between the temperatures of 30–50 °C/5 min whereas it lost nearly all of its activity at 70 °C/5 min. E_a and Z values were found to be 196.8 kJmol⁻¹ (r² = 0.996) and 2.16 °C (r² = 0.995), respectively.     

Foaming and drying behaviour of ripe bananas

The foaming of ripe bananas and the forced air-drying characteristics of the resulting banana foam mats were studied. Fresh banana puree with a density of 0.93 g/ml was foamed to a density of 0.50 g/ml after 12 min of whipping by the addition of 10 g/100 g soy protein as a foam inducer. Glyceryl monostearate did not induce foaming while commercially available food ingredients, Dream Whip and Gelatine induced foaming but such foams were not suitable for subsequent drying. Banana foam mats were dried at temperatures from 45°C to 90°C in a forced air, cabinet dryer, to a hard, porous and brittle solid which was amenable to grinding so as to produce a dehydrated banana powder. The transient drying behaviour of such mats was described by a capillary model of the form ln(M/M₀)=−Kt and over the bulk of the moisture ratio range, 0.05?M/M₀?1.0. The drying time (t) was directly related to the thickness of the foam mats. K values increased from 0.248 to 0.809 h⁻¹ as the drying air temperature was raised from 45°C to 90°C. Increasing the air velocity from 0.62 to 1.03 m/s did not profoundly influence the drying rate.     

Inhibition of polyphenoloxidase from apple by Maillard reaction products prepared from glucose or fructose with l-cysteine under various conditions of pH and temperature

The effects of Maillard reaction products (MRPs), synthesized from equimolar glucose or fructose with l-cysteine (1 mol l⁻¹) aqueous model mixtures, by modulating pH and temperature of heating, according to a two-factor and five-level experimental design, were investigated on polyphenoloxidase (PPO) activity from apple. Final pH and absorbance measurements at 350 nm were also selected as indicators of the Maillard reaction development and checked. In general, inhibitory potency (IP) of the mixtures increased with the increase in temperature (80-120°C) and the decrease in pH (pH 2.0-12.0) of the reaction medium. A linear relationship between the IP and heating time (0-48 h) or Abs.350 nm (0-70 AU) was demonstrated for glucose/cysteine system heated from 80°C to 120°C. Polarographic and spectrophotometric data were used to calculate kinetic constants and activation energy (Ea) values of inhibitory MRPs formation versus PPO activity and of those compounds absorbing at 350 nm. Ea values for these reactions were close, being 191 and 124 kJ mol⁻¹, respectively. The experimental design allowed to conclude that linear effects of both factors as well as a quadratic effect of pH were significant, leading to optimum conditions for the production of glucose-derived MRPs inhibitors. In most cases, glucose produced MRPs with higher IP compared to counterpart fructose-cysteine MRPs.     

Characterization of polyphenoloxidase (PPO) and total phenolic contents in medlar (Mespilus germanica L.) fruit during ripening and over ripening

Characterization of polyphenoloxidase (PPO) enzyme and determination of total phenolic concentrations during fruit ripening and over ripening in medlar (Mespilus germanica L.) were determined. During ripening, PPO substrate specificity, optimum pH and temperature, optimum enzyme and substrate concentrations were determined. Among the five mono- and di-phenolic substrates examined ((p-hydroxyphenyl) propionic acid, l-3,4-dihydroxyphenylalanine, catechol, 4-methylcatechol and tyrosine), 4-methylcatechol was selected as the best substrate for all ripening stages. A range of pH 3.0–9.0 was also tested and the highest enzyme activity was at pH 7.0 throughout ripening. The optimum temperature for each ripening stage was determined by measuring the enzyme activity at various temperatures over the range of 10–70 °C with 10 °C increments. The optimum temperatures were found to be 30, 20 and 30 °C, respectively, for each ripening stage. Optimum enzyme and substrate concentrations were found to be 0.1 mg/ml and 40 mM, respectively. The V_max and K_m value of the reaction were determined during ripening and found to be 476 U/mg protein and 26 mM at 193 DAFB (days after full bloom) – stage 1, 256 U/mg protein and 12 mM at 207 DAFB – stage 2, 222 U/mg protein and 8 mM at 214 DAFB – stage 3. For all ripening stages sodium metabisulfite markedly inhibited PPO activity. For stage 1 of ripening, Cu²⁺, Hg²⁺ and Al³⁺, for stage 2, Cu²⁺ and Hg²⁺, and for stage 3, Cu²⁺, Hg²⁺, Al³⁺ and Ca²⁺ strongly inhibited diphenolase activity. Accordingly, it can be concluded that as medlar fruit ripen there is no significant changes in the optimum values of polyphenoloxidases, although their kinetic parametres change. As the fruit ripening progressed through ripe to over-ripe, in contrary to polyphenoloxidase activity, there was an apparent gradual decrease in total fruit phenolic concentrations, as determined by using the aqueous solvents and water extractions.     

Hydrolysis of sucrose by invertase immobilized on nylon-6 microbeads

A commercial extracellular invertase (EC 3.2.1.26) from Saccharomyces cerevisiae has been inmobilized by covalent bonding on novel microbeads of nylon-6 using glutaraldehyde. The enzyme was strongly bound on the support, immobilized with an efficiency factor of 0.93. The biocatalyst showed a maximum enzyme activity when immobilized at pH 5.0, but optimum pH activity for both immobilized and free invertases was 5.5. The optimum temperatures for immobilized and free enzymes were 60 and 65 °C, respectively. Kinetic parameters were determined for immobilized and free invertases: V_max values were 1.37 and 1.06 mmol min⁻¹ mg⁻¹, respectively. The K_m and K_i values were 0.029 and 0.71 M for immobilized invertase and 0.024 and 0.69 M for free invertase. It was found that the thermal stability of the immobilized invertase with regard to the free one increased by 25% at 50 °C, 38% at 60 °C and 750% at 70 °C. The immobilized biocatalyst was tested in a tubular fixed-bed reactor to investigate its possible application for continuous sucrose hydrolysis. The effects of two different sugar concentrations and three flow rates on the productivity of the reactor and on the specific productivity of the biocatalyst were studied. The system demonstrated a very good productivity up to 2.0 M sugar concentration, with conversion factors of 0.95 and 0.97, depending on sucrose concentration in the feeding. This approach may serve as a simple technique and can be a feasible alternative to continuous sucrose hydrolysis in a fixed bed reactor for the preparation of fructose-rich syrup.     

Thermal properties and resistant starch content of green banana flour (Musa cavendishii) produced at different drying conditions

The objective of this research was to verify the effect of drying conditions on thermal properties and resistant starch content of green banana flour (Musa cavendishii). The green banana flour is a complex-carbohydrates source, mainly of resistant starch, and quantifying its gelatinization is important to understand how it affects food processing and the functional properties of the flour. The green banana flour was obtained by drying unripe peeled bananas (first stage of ripening) in a dryer tunnel at 52 °C, 55 °C and 58 °C and air velocity at 0.6 m s⁻¹, 1.0 m s⁻¹ and 1.4 m s⁻¹. The results obtained from differential scanning calorimetry (DSC) curves show a single endothermic transition and a flow of maximum heating at peak temperatures from (67.95 ± 0.31) °C to (68.63 ± 0.28) °C. ANOVA shows that only drying temperature influenced significantly (P < 0.05) the gelatinization peak temperature (Tp). Gelatinization enthalpy (ΔH) varied from 9.04 J g⁻¹ to 11.63 J g⁻¹ and no significant difference was observed for either temperature or air velocity. The resistant starch content of the flour produced varied from (40.9 ± 0.4) g/100 g to (58.5 ± 5.4) g/100 g, on dry basis (d. b.), and was influenced by the combination of drying conditions: flour produced at 55 °C/1.4 m s⁻¹ and 55 °C/1.0 m s⁻¹ presented higher content of resistant starch.     

Biochemical properties and potential endogenous substrates of polyphenoloxidase from chufa (Eleocharis tuberosa) corms

Polyphenoloxidase (PPO) was partially purified from chufa corms through ammonium sulphate precipitation and dialysis. Biochemical properties of chufa PPO were analysed using exogenous substrate catechol. Optimal pH and temperature for PPO activity were 5 and 45 °C. Ethylenediaminetetraacetic acid disodium salt and l-cysteine could not inhibit the PPO activity. However, sodium thiosulphate pentahydrate exhibited the strongest inhibiting effect, followed by ascorbic acid and anhydrous sodium sulphite. Except for K⁺, other metal ions such as Zn²⁺, Cu²⁺, Fe³⁺, Ca²⁺, Fe²⁺ and Na⁺ accelerated the enzymatic reaction between catechol and PPO. Kinetic analysis showed that the apparent K_m and V_max values were around 10.77 mM and 82 units/ml min. In addition, (−)-gallocatechin gallate, (−)-epicatechin gallate and (+)-catechin gallate isolated and identified from chufa corms were supposed to be the potential endogenous PPO substrates due to their ortho-diphenolic or pyrogallolic structures. These polyphenols might be catalysed by PPO, resulting in the browning of chufa corms after fresh-cut processing.     

Effects of light,temperature and water activity on the kinetics of lipoxidation in almond-based products

Lipoxidation in almond-derived products was investigated using the chemiluminescence (CL) and thiobarbituric acid-reactive substances (TBARS) methods to detect the first and later reaction products, respectively. The effects of light during storage at 5 °C, 22 °C and 40 °C were studied, as well as the effects of combined heat/water activity treatments in the 60–120 °C and 0.38–0.72 range. During storage, light was found to enhance the CL and TBARS values, and specific responses were observed in almond paste and the final Calisson product. During the heating of almond paste, as the initial water activity (a_w) increased, the CL rate constants increased during heating to 60 °C and 80 °C, but interestingly, these values decreased during further heating to 120 °C, whereas the maximum TBARS rate constants occurred at a_w 0.57 at all the heating temperatures tested. The activation energies, based on the CL and TBARS values, decreased specifically when the a_w increased from 0.38 to 0.72, giving overall values ranging from110 kJ mol⁻¹ to 60 kJ mol⁻¹. Likewise, in the same water activity range, the temperature-dependent rate constant enhancing factor (Q₁₀) decreased from 3.3 to 1.6.     

Purification and characterization of olive (Olea europaea L.) peroxidase – Evidence for the occurrence of a pectin binding peroxidase

Peroxidase from olive fruit (Olea europaea L., cv Douro) in a black ripening stage was purified to electrophoretic homogeneity, resulting in four cationic and four anionic fractions. The anionic fractions accounted for 92% of recovered activity and showed molecular masses of 18–20 kDa. The anionic fraction PODa4, the predominant fraction that comprised about 70% of total recovered activity, showed an isoelectric point of 4.4 and optimum pH and temperature of, respectively, 7.0 and 34.7 °C, and apparent K_m values of 41.0 and 0.53 mM, for phenol and H₂O₂, respectively. From the activity-temperature profile, the denaturation temperature and the changes in enthalpy and heat capacity for unfolding of PODa4 were estimated as being, respectively, 36.5 °C, 411.2 and −13.6 kJ mol⁻¹ K⁻¹. The activation energy for phenol oxidation by PODa4 was 99.1 kJ mol⁻¹, corresponding to a calculated temperature coefficient (Q₁₀) of 4. The arabinose (39 mol%) and galacturonic acid (38 mol%) content of the carbohydrate moiety indicated the existence of pectic material in the purified PODa4 fraction. Co-migration of the carbohydrate with the protein band in the isoelectric focusing electrophoresis, points to PODa4 fraction as being a pectin type binding peroxidase.     

Release kinetics of nisin from biodegradable poly(butylene adipate-co-terephthalate) films into water

The release kinetics of nisin from poly(butylene adipate-co-terephthalate) (PBAT) to distilled water was studied at of 5.6, 22 and 40 °C. The release kinetics of nisin from PBAT film was described using Fick’s second law of diffusion, partition coefficient, and Weibull model. The diffusion coefficients (D) determined were 0.93, 2.29, and 5.78 × 10⁻¹⁰ cm²/s at 5.6, 22, and 40 °C, respectively. The partition coefficients (K) calculated were 0.84, 3.89, and 5.2 × 10³ at 5.6, 22, and 40 °C, respectively. The nisin release data at selected temperatures were fitted with the Weibull model (R² > 0.97) with b and n values ranging from 0.02 to 0.98 and from 0.28 to 0.45, respectively. The temperature dependence of D, K, and Weibull model parameter b was modeled using the Arrhenius equation giving values of activation energy (E_a) of 38.3 kJ mol⁻¹ (for D), 38.5 kJ mol⁻¹ (for K), and 79.5 kJ mol⁻¹ (for b).     

Effects of ageing and low internal temperature of grilling on the formation of heterocyclic amines in beef Longissimus dorsi muscle

The mutagenic heterocyclic amines (HAs) originate in processed proteinaceous food. The effects of ageing (non-aged - i.e. 24 h post mortem vs. 14 and 28 days post mortem kept at 1 °C) and final internal temperature on cooking (T_i, 65 and 80 °C) on the content of HAs in grilled steaks (two-plated grill, temperature of 220 °C) were studied. HA precursors (creatine, creatinine, and free amino acids) and ageing indicators, such as instrumentally measured colour values, pH_ultimate values and length of myofibrilar fragments on raw and cutting values on grilled beef Longissimus dorsi muscles were determined. The muscles originated from eight commercially slaughtered Simmental bulls, 19-20 months old. The content of HAs was determined by a solid-phase extraction procedure. Meat ageing is accompanied by large changes in the chemical composition and structure of muscle tissues. In general, all the ageing indicators and precursors of HAs were influenced by ageing time at the 5% level or less. Creatine content declined significantly (non-aged: 6.00 mg g⁻¹, 14 days: 5.82 mg g⁻¹, and 28 days: 5.55 mg g⁻¹) and creatinine increased with days of ageing (non-aged: 0.19 mg g⁻¹, 14 days: 0.24 mg g⁻¹, and 28 days: 0.26 mg g⁻¹). Higher contents of total free amino acids were determined after 14 and 28 days of storage (28.18 μmol g⁻¹ and 37.59 μmol g⁻¹) than in non-aged beef (19.00 μmol g⁻¹). In this study, two HAs were determined: MeIQx (2-amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline) and PhIP (2—amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine). The content of HAs increases with ageing. At lower T_i, more MeIQx was formed; at higher T_i, more PhIP was formed. MeIQx was present in all samples while PhIP was found only in samples grilled to higher T_i. Samples treated to T_i = 80 °C generally contained less HAs (non-aged meat: 0.20 ng g⁻¹, 14 days: 0.26 ng g⁻¹, and 28 days: 0.28 ng g⁻¹) than samples treated to T_i = 65 °C (non-aged meat: 0.19 ng g⁻¹, 14 days: 0.36 ng g⁻¹, and 28 days: 0.39 ng g⁻¹) on account of MeIQx thermolability.     

Design and characterisation of an enzyme system for inulin hydrolysis

The optimal conditions for inulin hydrolysis using a commercial inulinase preparation, either free or immobilised in activated Amberlite were established by factorial design and surface response methodology. The immobilised biocatalyst displayed highest activity at pH 5.5 and 50 °C, whereas the optimum pH for the free form was slightly more acidic (4.5), and the optimum temperature was a little higher (55 °C). The model system estimated optimal pH and temperature values of 5.4 and 52 °C for the immobilised system and 4.9 and 56 °C for the free system. Michaelis–Menten type kinetics adequately described both free and immobilised bioconversion systems, which were evaluated under the respective optimal pH and temperature conditions. The use of a non-linear regression method for the determination of the kinetic parameters provided a best fit to the experimental data, as compared to a conventional Lineweaver–Burk linearisation. The K_m for inulin of the free biocatalyst was 153 g l⁻¹ at 55 °C and pH 4.5, whereas the apparent K_m for inulin of the immobilised biocatalyst was 108 g l⁻¹ at pH 5.5 and 50 °C. The reutilisation of the immobilised biocatalyst throughout consecutive batches was evaluated. A significant decrease of enzyme activity was observed in the first two batches, after which the system exhibited significant stability. The low cost of the support, the stability of the immobilised biocatalyst towards pH and temperature and its high affinity for the substrate suggests its potential for inulin hydrolysis.     

A β-galactosidase from chick pea (Cicer arietinum) seeds: Its purification,biochemical properties and industrial applications

A β-galactosidase from Cicer arietinum seeds has been purified to apparent electrophoretic homogeneity using a combination of various fractionation and chromatographic techniques, giving a final specific activity of 220 units mg⁻¹, with approximately 1840 fold purification. Analysis of the protein by SDS–PAGE revealed two subunits with molecular masses of 48 and 38 kDa, respectively. These bands were further confirmed with LC–MS/MS, indicating that Chick pea β-galactosidase (CpGAL) is a heterodimer. Molecular mass was determined to be 85 kDa by Superose-12 FPLC column, which is in agreement with the molecular mass suggested by mass spectroscopy to be 83 kDa. The optimum pH of the enzyme was 2.8 and it hydrolysed o-nitrophenyl β-d galactopyranoside (ONPG) with a K_m value of 1.73 mM at 37 °C. The energy of activation (E_a) calculated in the range of 35 to 60 °C, using Arrhenius equation, was determined to be 11.32 kcal mol⁻¹. The enzyme could also hydrolyse lactose, with an optimum pH of 4.0 at 40 °C. K_m and E_a for lactose hydrolysis was found to be 10 mM and 10.57 kcal mol⁻¹, respectively. The enzyme was found to be comparatively thermostable showing maximum activity at 60 °C for both ONPG and lactose. Galactose was found to be the competitive inhibitor. β-Galactosidase also exhibited glycoproteineous properties when applied on Con-A Sepharose column. The enzyme was localised in germinated seeds with X-gal activity staining and shown to be expressed prominently at grown radical tip and seed coat. Sequence alignment of CpGAL with other known plant β-galactosidase showed high amino acid sequence homology.     

Purification and characterisation of a polyphenol oxidase from Boletus erythropus and investigation of its catalytic efficiency in selected organic solvents

Polyphenol oxidase (PPO) was purified from Boletus erythropus using a Sepharose 4B-L-tyrosine-p-amino benzoic acid affinity column. Optimum pH and temperature were found to be 8.0 and 20 °C, respectively, using 4-methylcatechol as a substrate. The enzyme was extremely stable between pH 3.0 and 9.0 after 24 h incubation at 4 °C. B. erythropus PPO was also quite stable between 10 and 30 °C after 4 h incubation. The K_m and V_max values were calculated as 2.8 mM and 1430 U/mg protein by Lineweaver–Burk curve, respectively. The enzyme activity was inhibited by sodium metabisulfite, ascorbic acid, sodium azide and benzoic acid. It was seen that the mushroom PPO was an effective biocatalyst in selected organic solvents, such as dichloromethane, dichloroethane and toluene, when catechin was used as a substrate. All data support that B. erythropus has a highly active PPO, possessing similar biochemical and kinetic characteristics to other plant PPOs.