MACERATION ACTIVITY OF AN ENDOPOLYGALACTURONASE FROM CANDIDA MACEDONIENSIS

The yeast Candida macedoniensis produces constitutively an extracellular pectinolytic enzyme with a high maceration activity; the culture filtrate is free from foreign enzyme activity. The enzyme formation is optimal under strictly anaerobic conditions with N₂ gassing. The culture medium for optimal enzyme recovery is a nutrient solution containing 1% yeast extract, 2% peptone and 10% sucrose, at pH 3, 28°C. Characterization of the enzyme showed it to be an endopolygalacturonase. The pH and temperature optima of the enzyme differ for pectic acid cleavage and maceration activity, these being 4.5 and 50—53°C and 2.5 and 40°C, respectively. The enzyme activities could not be separated from one another by protein chemical methods (analytical and preparative isoelectric focussing). Dialysis of the enzyme-containing culture filtrate did not decrease the enzyme activity. The endopolygalacturonase from Candida macedoniensis leads to the release of plant cells from the tissue, without destroying the cells by lysing the cell walls. After two hours incubation of the substrate (carrot slices), the tissue mass consisted of cell clumps of up to 15 cells.     

Evaluation of pectolytic activities of enological interest in industrial enzyme preparations

 The purpose of this research was to determine the activity of several pectolytic enzymes, such as pectin methyl esterase, endopolymethylgalacturonase, endopolygalacturonase, exopolymethylgalacturonase, endopolygalacturonase, pectin transeliminase and pectic acid transeliminase, from within preparations that are available on the Portuguese market. The selected preparations were as follows: 15 pectolytic enzyme preparations, one hemicellulolytic preparation and one glucanase preparation. The measurements of enzyme activity were carried out using model solutions with 50 mg·l^–1 SO₂, pH 3.2, 25°C, with and without 12% ethanol (v/v), and a 24-h incubation period. Quantitatively, the results proved to be heterogeneous, significantly varying from preparation to preparation: all pectolytic enzyme activities in the 17 preparations analysed in this study were significantly different. The presence of ethanol reduced the enzymatic activity, apart from that of endopolymethylgalacturonase, and, in general, an increase of enzyme concentration raised the pectolytic enzyme activity. Received: 17 March 1997 / Revised version: 11 June 1997     

Evaluation of pectolytic activities of enological interest in industrial enzyme preparations

 The purpose of this research was to determine the activity of several pectolytic enzymes, such as pectin methyl esterase, endopolymethylgalacturonase, endopolygalacturonase, exopolymethylgalacturonase, endopolygalacturonase, pectin transeliminase and pectic acid transeliminase, from within preparations that are available on the Portuguese market. The selected preparations were as follows: 15 pectolytic enzyme preparations, one hemicellulolytic preparation and one glucanase preparation. The measurements of enzyme activity were carried out using model solutions with 50 mg·l^–1 SO₂, pH 3.2, 25°C, with and without 12% ethanol (v/v), and a 24-h incubation period. Quantitatively, the results proved to be heterogeneous, significantly varying from preparation to preparation: all pectolytic enzyme activities in the 17 preparations analysed in this study were significantly different. The presence of ethanol reduced the enzymatic activity, apart from that of endopolymethylgalacturonase, and, in general, an increase of enzyme concentration raised the pectolytic enzyme activity. Received: 17 March 1997 / Revised version: 11 June 1997     

CHARACTERIZATION OF AN 'ENDOPOLYGALACTURONASE' OF THE YEAST KLUYVEROMYCES MARXIANUS

Analytic isoelectric focusing showed that the ‘endopolygalacturonase’ from Kluyveromyces marxianus consists of 21 multiple enzyme components. Neither changes in cultivation conditions, enzyme substrate or reaction conditions resulted in any quantitative or qualitative differences in the enzyme patterns. Two of these multiple enzyme forms, the main band, (IP = pH 5.8) which accounted for approximately 95% of the total activity, and an ‘acid-band’ (IP = pH 2.7), were purified by means of preparative isoelectric focusing and their molecular weights and amino acid compositions were determined. The molecular weight of the main band was established as 76,000 daltons (2 subunits of 47,900 and 28,100), The molecular weight of the ‘acid-band’ was 32,200. Both amino acid analyses and molecular weight determinations suggested that the proteins are chemically and physically different. The pH optimum was 4 for the pure enzyme on different pectin substrates, e.g., high molecular weight pectic acid, low molecular weight pectic acid and highly methylated pectins B and C. The temperature optimum obtained for pure enzyme with high or low molecular-weight pectic acid as substrate was 40° C. V_max and Km-value determination at different pH values with low molecular weight pectic acid as a substrate was used to identify the catalytically active groups at the active site. They were tentatively identified as an unprotonated α-carboxyl group and a protonated carboxyl group of aspartic acid.     

On the response under different thermal treatments of the thermostable endopolygalacturonase produced byRhizopus nigricans

The influence of temperature and thermal treatments on the endopolygalacturonase activity produced byRhizopus nigricans was studied. The optimum temperature was at 50 °C. The enzymatic preparation also showed the highest endo pattern at this temperature. This endopolygalacturonase showed high thermostability between 90–100 °C and a bimodal behaviour in relation to the thermal treatments, because the enzyme was strongly inactivated at 70 °C. When the enzyme was treated at 90 and 70 °C consecutively, the inactivation at 70 °C was smaller.

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Einfluß der verschiedenen thermischen Behandlungen der thermostabilen Endopolygalacturonase vonRhizopus nigricans

Zusammenfassung Es wurde der Einfluß der Temperatur und der thermischen Behandlungen auf die Aktivität der Endopolygalacturonase vonRhizopus nigricans studiert. Hierbei wurde eine optimale Endtemperatur von 50 °C erreicht, wobei das Enzym seinen maximalen Charakter als Endo-Enzym belegt. Diese Endopolygalacturonase zeigt die größte Thermostabilität zwischen 90 °–100 °C und ein bimodales Verhalten, wenn man bedenkt, daß das Enzym bei 70 °C stark inaktiv ist. Wenn das Enzym zweimal hintereinander bei 90 °C und 70 °C behandelt worden ist, hat dies die Konsequenz, daß die Inaktivität bei 70 °C weniger ist.

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In memory of Dr. José María Núñez (1959–1990)     

On the response under different thermal treatments of the thermostable endopolygalacturonase produced byRhizopus nigricans

The influence of temperature and thermal treatments on the endopolygalacturonase activity produced byRhizopus nigricans was studied. The optimum temperature was at 50 °C. The enzymatic preparation also showed the highest endo pattern at this temperature. This endopolygalacturonase showed high thermostability between 90–100 °C and a bimodal behaviour in relation to the thermal treatments, because the enzyme was strongly inactivated at 70 °C. When the enzyme was treated at 90 and 70 °C consecutively, the inactivation at 70 °C was smaller.     

ASPERGILLUS NIGER ENDOPOLYGALACTURONASE

ABSTRACT A purified endopolygalacuronase (endo PG) from Aspergillus niger has been characterized. The optimum temperature was 45°C and the optimum pH was 4.1–4.2 with a highest stability at pH 6. The purified enzyme was a typical endo PG: the enzyme hydrolyzes about 56.7% and 7% of the glycosidic linkages of polygalacturonic acid and pectin (DE = 72.8%), respectively, and a loss of 50% in viscosity of polygalacturnoic acid was reached when only 2.3% of the glycosidic linkages of the substrate were split. The products at the maximum hydrolysis were mono-, di- and trigalacturonic acids. Endo PG has a V_max of 239 μ mole reducing groups per min per mg of protein and a K_m of 0.44 mg per ml of polygalacutronic acid (DP_n= 44). Sodium, potassium and ammonium ions stimulated endo PG activity, while divalent ions, or ethylene — diamine – tetracetic acid had no effect. Pectinolytic enzymes (saponifying enzymes and depolymerases) are widespread in nature; they are produced by bacteria, fungi and higher plants (Rombouts and Pilnik 1972; MacMillan and Sheiman 1974). They are involved in natural processes, as fruit physiological changes (Pilnik and Voragen 1970) and they are responsible for the phytopathogenicity of many microorganisms (Rexova-Benkova and Markovic 1976). These enzymes are very useful for their applications in food processing. Commercial preparations of fungal pectinolytic enzymes are employed in the production of fruit and vegetable juices (Rombouts and Pilnik 1978). These preparations contain mainly pectinesterases (pectin-pectyl hydrolase, E.C. 3. 1. 1. 11 and endopolygalacturonase (poly (1, 4 α D galacturonide) glycanohydrolase, E.C. 3. 2. 1. 15); for this reason, special attention was paid to endopolygalacturonase (endo PG (ENDO, 1964a, 1964b, 1964c; Yamasaki et al. 1966). Purified endo PG was also used as a tool for specific degradations of pectic substances (Talmadge et al. 1973) or material containing galacturonic acids (Kikuchi and Yokotsuka, 1973; Kikuchi and Sugimoto, 1976). In a previous paper (Thibault and Mercier 1978) we have described a rapid method for the purification of endo PG from a commercial preparation of pectolytic enzymes. This method is based on a remark of English et al. (1972) who observed that endo PG was delayed during chromatography on agarose gel (Bio-Gel A 0.15 m). We demonstrated that dialyzed endo PG from a crude preparation can be optimally bound by ion-exchange (Thibault 1978) to the agarose gel (Sepharose 6B) when the column is eluted at pH 4.3–4.4 (20 mM acetate buffer). The endo PG released by a 0–0.15 M NaCl gradient is therefore purified 62 times if the activity is determined by a viscosimetric method and 36 times if the activity is expressed in n. katals (cf. enzyme assay methods for the definitions). The present paper reports characterization and some physico-chemical properties of the purified enzyme.     

PURIFICATION OF PEACH POLYPHENOL OXIDASE IN THE PRESENCE OF ADDED PROTEASE INHIBITORS1

Crude preparations of peach fruit (Prunus persica Batsch cv. Redskin) polyphenol oxidase (PPO) showed many apparent isoenzyme forms. Some of these forms were probably the result of proteolytic action of peach proteases while other forms were the result of association of PPO with carbohydrate materials. In the presence of protease inhibitors, Trasylol and phenylmethylsul-fonyl fluoride, three apparent isoenzyme forms of PPO were purified to homogeneity. The purification scheme included hydrophobic chromatography on phenyl sepharose CL-4B, hydroxylapatite chromatography, DEAE cellulose chromatography, and gel filtration on Ultrogel AcA 34. Minor contaminants remaining after these steps were separated from PPO by gel electrophoresis. The major PPO isoenzyme form (A) was purified 44 fold with an overall yield of 5.6% and contained no detectable carbohydrates. Isoenzyme forms A' and A' were purified 104 and 67 fold respectively, but still were associated with carbohydrate material. Cesium chloride centrifugation partially removed the carbohydrates associated with PPO A' and A'. Purified peach PPO A showed greater activity toward D-catechin (539%) and pyrogallol(l82%) than to catechol (100%). An apparent K₃ of 4, 0.3, and 2 mM was obtained with D-catechin, pyrogallol and catechol, respectively. The enzyme was severely inhibited by 10 μM 2,3-naphthalenediol (91%) and by 10 pM diethyl dithiocarbamate (100%).     

Optimizing the Enzymatic Maceration of Foliole Purée from Hard Pieces of Hearts of Palm (Euterpe edulis Mart.) using Response Surface Analysis

Response surface analysis was applied to investigate modifications in viscosity of foliole purée treated with commercial enzyme preparations high in cellulase and endopolygalacturonase (pectinase) with changes in enzyme concentration (0.3–1.2%), incubation time (3.3–6.7 hr) and temperature (40–50°C). Foliole purée was obtained by trituration of hard pieces of hearts of palm (Euterpe edulis) and incubated in a rotating agitator. After treatment with 0.81% cellulase for ～5 hr at 50°C, a fourfold reduction in viscosity was found relative to a control sample. The minimum viscosity for this treatment was within the experimental range investigated. Optimized experimental conditions for treatment with pectinase however were outside the experimental range. The application of the cellulase preparation to the purée resulted in a 10% increase in yield of edible palm.     

Use of contact prints for recording polyphenoloxidase isoenzymes separated by electrophoresis

Crude extracts of polyphenoloxidase (PPO) (E.C. 1.14.18.1) from a variety of food and plant sources were subjected to polyacrylamide gel electrophoresis. Following electrophoresis, the gels were stained for enzyme activity using catechol and L-dopa as substrates. Contact prints were made of the enzyme stained gels 30 min after enzyme staining and on the following day. The negative images (white bands on a dark background) clearly showed the number, type, mobility and variation in enzyme staining of isoenzyme forms of PPO separated by electrophoresis. Contact prints were used to identify PPO isoenzyme forms in the leaves, stipules, roots, and stems of broad beans. This method was also used to demonstrate that a single isoenzyme form of PPO in broad bean leaves decreased with leaf age. Photographs, using professional rapid process X-ray copy film, were also taken of enzyme stained gels for comparison to contact prints. Using contact prints, permanent records of PPO isoenzyme forms can be obtained rapidly, are inexpensive, and reproduce actual gel patterns.     

Comparative studies of pyruvate kinase from PSE and normal pig muscles

A fast breakdown of glycogen is observed in muscles of stress-susceptible pigs leading to pale, soft and exudative (PSE) meat. We report a comparative study of pyruvate kinase from muscles of normal and PSE-prone pigs. Compared with the enzyme from normal muscle, pyruvate kinase isolated from PSE muscle shows a five times lower Michaelis constant,K _m, for phosphoenol pyruvate and a more than ten times higherk _cat/K _m value. The pH dependency of the enzymatic activity is shifted to more acidic values for pyruvate kinase from PSE muscles. According to isoelectric focusing, pyruvate kinase from PSE muscle consists of three isoforms, while only two isoforms are detectable in pyruvate kinase preparations from normal pigs. The various isoforms were isolated by preparative isoelectric focusing and their steady-state properties were compared. Isoform 3, which is found only in PSE muscle, shows a 10-fold higher specific activity, a 30-fold lowerK _m value and a 100-fold increasedk _cat/K _m value for phosphoenol pyruvate as compared to isoform 1. The presence of isoform 3 in PSE muscle appears to be responsible for the high activity of this enzyme under the more acidic conditions prevailing in PSE muscle. In vitro phosphorylation and dephosphorylation experiments using total enzyme and purified isoenzyme 1 suggest that isoforms 2 and 3 arise from isoform 1 by phosphorylation. Thus protein phosphorylation seems to be responsible for the shift in activity of pyruvate kinase, a key enzyme of glycolysis, under the acidic conditions of PSE muscles.     

Thermal Inactivation Kinetics of Peroxidase in Coriander Leaves

Design of efficient blanching treatments requires knowledge of critical factors such as enzyme inactivation kinetic parameters and relative proportions of heat-labile and heat-resistant fractions, which is unique in each vegetable. Thermal inactivation curves for peroxidase in coriander leaves were determined in the temperature range of 70 to 100 °C and in steam. The isothermal data were statistically treated using both linear and nonlinear regression. Applicability of various enzyme inactivation models available in the literature was critically evaluated. The two-fraction first-order model was found to be the best model to describe the peroxidase inactivation kinetics in coriander leaves (R ² > 0.97). Kinetic parameters were determined for heat-labile and heat-resistant isoenzyme fractions. The temperature dependence of the rate parameters in the present study did not follow the Arrhenius relationship.     

Polyphenoloxidase activity from strawberry fruit (Fragaria ananassa,Duch., cv Selva): characterisation and partial purification

In this work, polyphenoloxidase (PPO) from Selva strawberry fruit (Fragaria × ananassa, Duch) was extracted, characterised and partially purified. The activity of PPO was analysed in crude extracts obtained from either fresh fruits or acetone powder. The presence of NaCl and Triton X‐100 in the extraction buffer caused a marked increase in enzyme extractability. The enzyme showed an apparent K_m value of 11.2 mM with pyrocatechol as substrate. The maximum enzyme activity was observed at 50 °C and pH 5.3–6.0 without SDS and pH 7.2 in the presence of SDS. The presence of SDS increased PPO activity at pH 7.2 but diminished it at pH 6.0. The enzyme showed high thermal stability and maintained activities equal to or greater than 50% of its maximum activity in the 2.6–9.3 pH range. One polyphenoloxidase isoenzyme was detected in crude extracts of all ripening stages, showing an isoelectric point of 7.3. The specific activity of PPO decreased continuously through fruit ripening. Maximum specific activities were found at the ‘small green’ and ‘large green’ ripening stages. A total enzyme extract was partially purified by means of (NH₄)₂SO₄ precipitation and cationic exchange chromatography in an FPLC system. The purification grade achieved was near 25. The partially purified enzyme showed an isoelectric point equal to 7.3 and a molecular mass of 135 ± 4 kDa for the native protein. © 2000 Society of Chemical Industry     

Polygalacturonase isoenzymes of fungi involved in the breakdown of sulphited strawberries

The polygalacturonases of four fungi, Rhizopus stolonifer, Rhizopus sexualis, Mucor piriformis and Aureobasidium pullulans, suspected of involvement in the breakdown of sulphited strawberries, have been resolved into a number of distinct enzyme forms. These isoenzymes differ from each other with respect to isoelectric points, molecular weights, resistance to extremes of pH and temperature, and susceptibility to inhibitors. The isoenzymes produced by any one organism in culture were identical to those produced in inoculated strawberries, although the relative proportions of the different forms was altered. Ten separate samples of microbially contaminated fruit from commercial sources, all of which subsequently suffered disintegration during storage in sulphite liquor, were analysed for polygalacturonase activity. The main isoenzyme present in each batch had an isoelectric point close to 7.1. Comparisons between the properties of polygalacturonases from field sources and those of known origin, revealed that Rhizopus sexualis was the most likely source organism for nine of the ten enzymes, Rhizopus stolonifer probably being responsible for the tenth. The polygalacturonase of isoelectric point near 7.1 was much more effective at macerating sulphited strawberries than the other isoenzymes tested. It also retained activity considerably longer than the others when added to sulphite liquor. It is concluded that a single polygalacturonase isoenzyme secreted by Rhizopus sexualis was responsible for disintegration of all but one of the strawberry samples examined.     

Purification and Characterization of Polyphenol Oxidase from Akko XIII Loquat (Eriobotrya japonica cv Akko XIII)

Akko XIII is an important loquat variety grown in Turkey. As with many fruits and vegetables, enzymatic browning catalyzed by polyphenol oxidase (PPO) also occurs in loquats. PPO from Akko XIII loquat was extracted and purified through (NH₄)₂SO₄ precipitation, dialysis and ion exchange chromatography. The enzyme showed several peaks with PPO activity on DEAE-Toyopearl 650 M column, of which only two (isoenzyme A and isoenzyme B) were characterized. Assay of activity of the isoenzymes between pH 3.04 and 7.80 using catechol as substrate showed two activity peaks, one at acidic pH and the other at neutral pH. pH optima of isoenzyme A and B were found to be at 7.4 and 4.98, respectively. The Km values of isoenzyme A and B using catechol as substrate were found to be 152.3 mM and 5.4 mM, respectively. They both displayed maximal activity at 30^oC. The two isoenzymes displayed different heat resistance and sensitivity towards various inhibitors.     

Polyphenoloxidase from DeChaunac grapes

Polyphenoloxidase (PPO) from red grape cultivar, DeChaunac, grown in New York State was isolated and purified 17-fold by using Phenyl Sepharose CL-4B column. Disc gel electrophoresis revealed near homogeneity of three isoenzyme bands. The molecular weight of this enzyme ranged from 73 000 to 85 000. The temperature and pH optima of the purified enzyme were 20 °C and 6.0, respectively. Kinetic studies showed that the thermal inactivation of the PPO followed first-order kinetics, with the activation energy, Ea = 52.39 Kcal mol^?1. The substrate specificity showed a high degree of PPO activity toward o-diphenolic compounds with the highest affinity toward caffeic acid among substrates studied. The apparent K_m values for caffeic acid and 4-methylcatechol as substrates for the Dechaunac PPO were 16 mmol and 25 mmol, respectively. The most potent inhibitors of the PPO were D.L-dithiothreitol and sodium metasulphite at the concentration level of 0.5 mmol.     

CLASSIFICATION OF GREEN PEA PEROXIDASES BY PREPARATIVE ISOELECTRIC FOCUSING

Green pea peroxidase isoenzymes were partially purified, identified, and grouped by preparative isoelectric focusing in granulated gel (PEGG), using a modified cascade method. Isoenzyme patterns were detected utilizing a substrate-impregnated paper print technique. Direct application of crude extract on PEGG proved to be a valuable tool for partial purification of peroxidase isoenzymes. A substrate-impregnated paper print technique enabled the detection of isoenzyme patterns and may replace the time-consuming conventional peroxidase activity assay. Twelve isoenzymes (pIs ranging from 5.5–8.6), isolated on analytical thin-layer isoelectric focusing on polyacrylamide gel, were isoelectrically homogeneous.     

Identification of Tyrosinase in Mushrooms by Isoelectric Focusing

Isoenzymes of tyrosinase were identified in mushrooms using vertical polyacrylamide gel isoelectric focusing in conjunction with a pH 4-7 gradient. The M2, M8, and M9 strains of Agaricus could be distinguished by differences in lower pI isoenzyme forms around pH 4. In the M8 strain, the epidermis, cap flesh, gill, and stalk tissue showed similar isoenzyme after isoelectric focusing, but the distribution of staining intensity appeared different for each type tissue. Younger developmental stages of M8 mushrooms showed a different isoenzyme profile than older ones. Mature mushrooms cut at the stipe showed a different distribution of isoenzyme staining than those not cut at the stipe, indicating possible activation of latent enzyme or new synthesis of specific tyrosinase isoenzymes.     

Characteristics and Novel Features of Thermostable α-Amylase Produced by Bacillus licheniformis M27 under Solid State Fermentation

The purified α-amylase from Bacillus licheniformis M27, produced under solid state fermentation technique, showed novel characteristics as compared to those reported by other workers for the purified α-amylases from B. licheniformis obtained by submerged fermentation process. Some of the novel features of the characteristics of the enzyme from B. licheniformis M27 include two peaks for pH optima at 6.5–7.0 and 8.5–9.0, gradual loss of activity to about 86% between pH 7.0–7.5 followed by rise to full activity between 7.5–8.5, temperature optimum at 85–90°C at pH 7.0 and 9.0, sharp fall in stability at acidic pH values and the thermostability response which is more similar to the enzyme from other species of Bacillus. The moleculuar weight of the enzyme was found to be 19,500 ± 500 and 56,000 ± 2,000 when determined by gel filtration and SDS PAGE, respectively. The activation energy is 20.4 times lower than that reported for the enzyme from another strain of B. licheniformis. It also showed differences in the contents of amino acids such as serine, proline and methionine.     

Penicillium commune, P. camembertii, the origin of white cheese moulds, and the production of cyclopiazonic acid

The taxonomy of Penicillia producing the mycotoxin cyclopiazonic acid, including isolates classified as Penicillium aurantiogriseum and P. puberulum, is reviewed on the basis of morphology, physiology, mycotoxin production and isoenzyme profiles. It is concluded that P. puberulum, as neotypified by Pitt in his 1979 monograph, is a synonym of P. aurantiogriseum. The correct name for saprophytic Penicillia producing cyclopiazonic acid is P. commune with P. palitans as a synonym. The moulds used in the manufacture of white cheeses, which are all classified in P. camembertii, and which also produce cyclopiazonic acid, are domesticated fungi derived from P. commune.