Partial purification,characterization, and thermal and high-pressure inactivation of pectin methylesterase from carrots (Daucus carrota L.)

Pectin methylesterase (PME) from carrots (Daucus carrota L.) was extracted and purified by affinity chromatography on a CNBr-Sepharose 4B-PME inhibitor column. A single protein and PME activity peak was obtained. A biochemical characterization in terms of molar mass (MM), isoelectric points (pI), and kinetic parameters of carrot PME was performed. In a second step, the thermal and high-pressure stability of the enzyme was studied. Isothermal and combined isothermal-isobaric inactivation of purified carrot PME could be described by a fractional-conversion model.     

Volatile flavor constituents of acerola (Malpighia emarginata DC.) fruit.

Volatile components were isolated from acerola fruit by simultaneous steam distillation-solvent extraction according to the Likens-Nickerson method and analyzed by GC and GC-MS methods. One hundred fifty constituents were identified in the aroma concentrate, from which furfural, hexadecanoic acid, 3-methyl-3-butenol, and limonene were found to be the major constituents. The amounts of esters, 3-methyl-3-butenol, and their various esters were thought to contribute to the unique flavor of the acerola fruit.     

Partial purification and characterization of polyphenol oxidase from banana (Musa sapientum L.) peel

Polyphenol oxidase (EC 1.10.3.1, o-diphenol: oxygen oxidoreductase, PPO) of banana (Musa sapientum L.) peel was partially purified about 460-fold with a recovery of 2.2% using dopamine as substrate. The enzyme showed a single peak on Toyopearl HW55-S chromatography. However, two bands were detected by staining with Coomassie brilliant blue on PAGE: one was very clear, and the other was faint. Molecular weight for purified PPO was estimated to be about 41 000 by gel filtration. The enzyme quickly oxidized dopamine, and its Km value (Michaelis constant) for dopamine was 3.9 mM. Optimum pH was 6.5 and the PPO activity was quite stable in the range of pH 5-11 for 48 h. The enzyme had an optimum temperature at 30 degrees C and was stable up to 60 degrees C after heat treatment for 30 min. The enzyme activity was strongly inhibited by sodium diethyldithiocarbamate, potassium cyanide, L-ascorbic acid, and cysteine at 1 mM. Under a low buffer capacity, the enzyme was also strongly inhibited by citric acid and acetic acid at 10 mM.     

Separation and characterization of a salt-dependent pectin methylesterase from Citrus sinensis var. Valencia fruit tissue

A pectin methylesterase (PME) from sweet orange fruit rag tissue, which does not destabilize citrus juice cloud, has been characterized. It is a salt-dependent PME (type II) and exhibits optimal activity between 0.1 and 0.2 M NaCl at pH 7.5. The pH optimum shifted to a more alkaline range as the salt molarity decreased (pH 8.5-9.5 at 50 mM NaCl). It has an apparent molecular mass of 32.4 kDa as determined by gel filtration chromatography, an apparent molecular mass of 33.5 kDa as determined by denaturing electrophoresis, and a pI of 10.1 and exhibits a single activity band after isoelectric focusing (IEF). It has a K(m) of 0.0487 mg/mL and a V(max) of 4.2378 nkat/mg of protein on 59% DE citrus pectin. Deblocking the N-terminus revealed a partial peptide composed of SVTPNV. De-esterification of non-calcium-sensitive pectin by 6.5% increased the calcium-sensitive pectin ratio (CSPR) from 0.045 +/- 0.011 to 0.829 +/- 0.033 but had little, if any, effect on pectin molecular weight. These properties indicate this enzyme will be useful for studying the PME mode of action as it relates to juice cloud destabilization.     

Cloning and expression of an acidic pectin methylesterase from jelly fig (Ficus awkeotsang)

Pectin methylesterase (PME) is the key enzyme responsible for the gelation of jelly curd in the water extract of jelly fig (Ficus awkeotasang) achenes. The jelly fig PME extracted from achenes was isoelectrofocused at pH 2.5 and subjected to N-terminal amino acid sequencing. A cDNA fragment encoding the mature protein of this acidic PME was obtained by PCR cloning using a poly(T) primer and a degenerate primer designed according to the N-terminal sequence of the purified PME. The complete cDNA sequence of its precursor protein was further obtained by PCR using the same strategy. The PME clone was overexpressed in Escherichia coli, and its expressed protein was immunologically recognized as strongly as the original antigen using antibodies against purified PME. Fractionation analysis revealed that the overexpressed PME was predominantly present in the pellet and thus presumably formed insoluble inclusion bodies in E. coli cells.     

Mungbean [Vigna radiata (L.) Wilczek] globulins: purification and characterization

Vicilin type (8S) and basic 7S globulins and legumin type (11S) globulins were isolated from mungbean [Vigna radiata (L.) Wilczek]. The native molecular weights of the different globulin types were 360000 for legumin, 200000 for vicilin, and 135000 for basic 7S. Some of the 8S globulin apparently complexed and coeluted with the 11S on gel filtration. On SDS-PAGE, 11S was composed of two bands of 40000 and 24000, 8S was composed of 60000, 48000, 32000, and 26000 bands, and basic 7S was composed of 28000 and 16000 bands. The percent composition of total globulins was estimated to be as follow: 8S, 89%; basic 7S, 3.4%; and 11S, 7.6%. The basic 7S and 11S but not the 8S globulins were found to have disulfide bonds. The presence of carbohydrates by conjugated peroxidase reaction was observed in all bands of 8S, the acidic polypeptide of basic 7S, and its complex but not in 11S. The 28000 basic 7S band and its 42000 complex and the first three major bands of 8S cross-reacted with antibodies to all types of soybean conglycinin subunits (alpha, alpha', and beta), whereas the fourth band cross-reacted only with the anti-beta subunit. None of the mungbean globulins cross-reacted with anti-soybean glycinin. Basic 7S was found to be easily extracted with 0.15 M NaCl, 11S was extracted with 0.35 M NaCl,and 8S was extracted over a wide range of NaCl concentrations. The N-terminal sequences of the different subunits/fragments of the globulins were determined and found to have strong homology with storage proteins of other legumes and crops.     

Characterization of a salt-independent pectin methylesterase purified from valencia orange peel

The pectin methylesterase (PME; EC 3.1.1.11) present in a commercial orange peel enzyme preparation was characterized to establish its identity among the multiple PME isozymes present in Valencia orange (Citrus sinensis L.) peel. We show the commercial enzyme corresponds to the major peak 2 PME previously separated by heparin-Sepharose chromatography (Cameron et al., J. Food Sci. 1998, 63, 253). Both PMEs have comparable elution profiles on cation-exchange and hydrophobic-interaction perfusion chromatography columns, molecular weights (ca. 34 kDa) and pI (pH 9.2), and biochemical properties, including a broad pH activity range and activity in the absence of added cations. An identical partial amino terminal peptide sequence was also obtained for the PMEs, which further demonstrated a structural identity with other plant PMEs. The biochemical and structural properties readily distinguish this Valencia orange PME from salt-dependent isozymes and further suggest that it is an ortholog to the salt-independent fruit-specific isozyme of tomato. This work provides a well-defined, enzymatically homogeneous, salt-independent (type 1) plant PME isozyme that is suitable for studying details of the enzyme's mode of action and for use in modifying methylester patterns for studying the structure-functional property relationships in pectin.     

Isolation, characterization, and pectin-modifying properties of a thermally tolerant pectin methylesterase from Citrus sinensis var. Valencia

The thermally tolerant pectin methylesterase (TT-PME) was isolated as a monocomponent enzyme from sweet orange fruit (Citrus sinensis var. Valencia). It was also isolated from flower and vegetative tissue. The apparent molecular weight of fruit TT-PME was 40800 by SDS-PAGE and the isoelectric point estimated as pI 9.31 by IEF-PAGE. MALDI-TOF MS identified no tryptic-peptide ions from TT-PME characteristic of previously described citrus PMEs. TT-PME did not absolutely require supplemented salt for activity, but salt activation and pH-dependent activity patterns were intermediate to those of thermolabile PMEs. Treatment of non-calcium-sensitive pectin with TT-PME (reducing the degree of methylesterification by 6%) increased the calcium-sensitive pectin ratio from 0.01 to 0.90, indicating a blockwise mode of action. TT-PME produced a significantly lower end-point degree of methylesterification at pH 7.5 than at pH 4.5. Extensive de-esterification with TT-PME did not reduce the pectin molecular weight or z-average radius of gyration, as determined by HPSEC.     

Characterization and purification of polyphenol oxidase from artichoke (Cynara scolymus L.)

In this study, the polyphenol oxidase (PPO) of artichoke (Cynara scolymus L.) was first purified by a combination of (NH(4))(2)SO(4) precipitation, dialysis, and a Sepharose 4B-L-tyrosine-p-aminobenzoic acid affinity column. At the end of purification, 43-fold purification was achieved. The purified enzyme migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis indicated that PPO had a 57 kDa molecular mass. Second, the contents of total phenolic and protein of artichoke head extracts were determined. The total phenolic content of artichoke head was determined spectrophotometrically according to the Folin-Ciocalteu procedure and was found to be 425 mg 100 g(-1) on a fresh weight basis. Protein content was determined according to Bradford method. Third, the effects of substrate specificity, pH, temperature, and heat inactivation were investigated on the activity of PPO purified from artichoke. The enzyme showed activity to 4-methylcatechol, pyrogallol, catechol, and L-dopa. No activity was detected toward L-tyrosine, resorsinol, and p-cresol. According to V(max)/K(m) values, 4-methylcatechol (1393 EU min(-1) mM(-1)) was the best substrate, followed by pyrogallol (1220 EU min(-1) mM(-1)), catechol (697 EU min(-1) mM(-1)), and L-dopa (102 EU min(-1) mM(-1)). The optimum pH values for PPO were 5.0, 8.0, and 7.0 using 4-methylcatechol, pyrogallol, and catechol as substrate, respectively. It was found that optimum temperatures were dependent on the substrates studied. The enzyme activity decreased due to heat denaturation of the enzyme with increasing temperature and inactivation time for 4-methylcatechol and pyrogallol substrates. However, all inactivation experiments for catechol showed that the activity of artichoke PPO increased with mild heating, reached a maximum, and then decreased with time. Finally, inhibition of artichoke PPO was investigated with inhibitors such as L-cysteine, EDTA, ascorbic acid, gallic acid, d,L-dithiothreitol, tropolone, glutathione, sodium azide, benzoic acid, salicylic acid, and 4-aminobenzoic acid using 4-methylcatechol, pyrogallol, and catechol as substrate. The presence of EDTA, 4-aminobenzoic acid, salicylic acid, gallic acid, and benzoic acid did not cause the inhibition of artichoke PPO. A competitive-type inhibition was obtained with sodium azide, L-cysteine, and d,L-dithiothreitol inhibitors using 4-methylcatechol as substrate; with L-cysteine, tropolone, d,L-dithiothreitol, ascorbic acid, and sodium azide inhibitors using pyrogallol as substrate; and with L-cysteine, tropolone, d,L-dithiotreitol, and ascorbic acid inhibitors using catechol as a substrate. A mixed-type inhibition was obtained with glutathione inhibitor using 4-methylcatechol as a substrate. A noncompetitive inhibition was obtained with tropolone and ascorbic acid inhibitors using 4-methylcatechol as substrate, with glutathione inhibitor using pyrogallol as substrate, and with glutathione and sodium azide inhibitors using catechol as substrate. From these results, it can be said that the most effective inhibitor for artichoke PPO is tropolone. Furthermore, it was found that the type of inhibition depended on the origin of the PPO studied and also on the substrate used.     

11S and 7S globulins of coconut (Cocos nucifera L.): purification and characterization

Total globulins extracted with 0.4 M NaCl in buffer from coconut endosperm separated into two peaks on gel filtration: peak I corresponding to 11S globulin or cocosin and peak II to 7S globulin with native molecular weights of 326 000 and 156 000, respectively. The percent composition of total globulins was estimated to be 11S, 86% and 7S, 14%. On SDS-PAGE, cocosin resolved into two closely migrating bands at approximately 34 000 (acidic polypeptide) and another set of 2 bands at 24 000 (basic polypeptide). Each set consisted of one darkly stained band and one lightly stained band. The 7S globulin consisted of three bands of 16 000, 22 000, and 24 000. Three isoforms of cocosin were identified after anion exchange chromatography. Cocosin, but not the 7S, was found to have disulfide bonds. Using periodic acid-Schiff's reagent, all of the bands of cocosin on SDS-PAGE were positive for carbohydrate. However, when con A-peroxidase was used, only the basic polypeptide stained positively for carbohydrate. For the 7S globulin, no carbohydrate group was detected using the PAS and con A-peroxidase tests. The 7S globulin was easily extracted with 0.10-0.15 M NaCl, whereas cocosin was extracted with 0.35 M NaCl. The N-terminal amino acid sequences of the 34 k band and 24 k band of cocosin were SVRSVNEFRXE and GLEETQ, respectively, and that of the 7S was EQEDPELQK.     

Activity and process stability of purified green pepper (Capsicum annuum) pectin methylesterase

Pectin methylesterase (PME) from green bell peppers (Capsicum annuum) was extracted and purified by affinity chromatography on a CNBr-Sepharose-PMEI column. A single protein peak with pectin methylesterase activity was observed. For the pepper PME, a biochemical characterization in terms of molar mass (MM), isoelectric points (pI), and kinetic parameters for activity and thermostability was performed. The optimum pH for PME activity at 22 degrees C was 7.5, and its optimum temperature at neutral pH was between 52.5 and 55.0 degrees C. The purified pepper PME required the presence of 0.13 M NaCl for optimum activity. Isothermal inactivation of purified pepper PME in 20 mM Tris buffer (pH 7.5) could be described by a fractional conversion model for lower temperatures (55-57 degrees C) and a biphasic model for higher temperatures (58-70 degrees C). The enzyme showed a stable behavior toward high-pressure/temperature treatments.     

Isolation,purification, and biochemical characterization of a novel water soluble protein from Inca peanut (Plukenetia volubilis L.)

A water soluble storage albumin from Inca peanut (IPA) accounted for approximately 25% (w/w) of defatted seed flour weight, representing 31% of the total seed protein. IPA is a 3S storage protein composed of two glycosylated polypeptides, with estimated molecular weights (MW) of 32800 and 34800 Da, respectively. IPA has an estimated sugar content of 4.8% +/- 0.92% (n = 6). IPA is a basic protein (pI of approximately 9.4) and contains all of the essential amino acids in adequate amounts when compared to the FAO/WHO recommended pattern for a human adult. The tryptophan content of IPA is unusually high (44 mg/g of protein), whereas the phenylalanine content is low (9 mg/g of protein). IPA is a highly digestible protein in vitro.     

Partial purification of polyphenol oxidase from Chinese cabbage Brassica rapa L

Polyphenol oxidase (PPO) was purified and characterized from Chinese cabbage by ammonium sulfate precipitation and DEAE-Toyopearl 650M column chromatography. Substrate staining of the crude protein extract showed the presence of three isozymic forms of this enzyme. The molecular weight of the purified enzyme was estimated to be approximately 65 kDa by gel filtration on Toyopearl HW-55F. On SDS-PAGE analysis, this enzyme was composed of a subunit molecular weight of 65 kDa. The optimum pH was 5.0, and this enzyme was stable at pH 6.0 but was unstable below pH 4.0 or above pH 7.0. The optimum temperature was 40 degrees C. Heat inactivation studies showed temperatures >40 degrees C resulted in loss of enzyme activity. PPO showed activity to catechol, pyrogallol, and dopamine (K(m) and V(max) values were 682.5 mM and 67.6 OD/min for catechol, 15.4 mM and 14.1 OD/min for pyrogallol, and 62.0 mM and 14.9 OD/min for dopamine, respectively). The most effective inhibitor was 2-mercaptoethanol, followed in decreasing order by ascorbic acid, glutathione, and L-cysteine. The enzyme activity of the preparation was maintained for 2 days at 4 degrees C but showed a sudden decreased after 3 days.     

Functional expression in pichia pastoris of an acidic pectin methylesterase from jelly fig (Ficus awkeotsang)

A cDNA fragment encoding an acidic pectin methylesterase (PME) of jelly fig achene was successfully expressed in Pichia pastoris under the control of the glyceraldehydes-3-phosphate dehydrogenase promoter. The recombinant PME was produced as a secretory protein by N-terminal fusion of a cleavable prepropeptide for signal trafficking, and thus easily harvested from the culture medium. Compared with native N-glycosylated PME (38 kDa) purified from jelly fig achenes, this recombinant PME (45 kDa) appeared to be hyperglycosylated. Activity staining indicated that the recombinant PME was functionally active. Yet the hyperglycosylated recombinant PME possessed thermostability and enzymatic capability over a broad pH range equivalent to those of the native PME. The success of functional production of this acidic jelly fig PME in P. pastoris has significantly broadened its applications in industry.     

Purification and characterization of polyphenol oxidase from cauliflower (Brassica oleracea L.)

Polyphenol oxidase (PPO) of cauliflower was purified to 282-fold with a recovery rate of 8.1%, using phloroglucinol as a substrate. The enzyme appeared as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The estimated molecular weight of the enzyme was 60 and 54 kDa by SDS-PAGE and gel filtration, respectively. The purified enzyme, called phloroglucinol oxidase (PhO), oxidized phloroglucinol (K(m) = 3.3 mM) and phloroglucinolcarboxylic acid. The enzyme also had peroxidase (POD) activity. At the final step, the activity of purified cauliflower POD was 110-fold with a recovery rate of 3.2%. The PhO and POD showed the highest activity at pH 8.0 and 4.0 and were stable in the pH range of 3.0-11.0 and 5.0-8.0 at 5 °C for 20 h, respectively. The optimum temperature was 55 °C for PhO and 20 °C for POD. The most effective inhibitor for PhO was sodium diethyldithiocarbamate at 10 mM (IC(50) = 0.64 and K(i) = 0.15 mM), and the most effective inhibitor for POD was potassium cyanide at 1.0 mM (IC(50) = 0.03 and K(i) = 29 μM).     

Partial purification and kinetic characterization of a carotenoid cleavage enzyme from quince fruit (Cydonia oblonga)

For the first time, a cytosolic carotenoid cleavage enzyme isolated from quince (Cydonia oblonga) fruit is described. The enzyme was partially purified by using centrifugation, acetone precipitation, ultrafiltration (300 kD, 50 kD), isoelectric focusing (pH 3-10), and sodium dodecyl sulfate polyacrylamide gel electrophoresis (7.5%). In this way, an enzymatically active protein fraction was obtained that contained three similar proteins, all exhibiting molecular weights in the range of 20 kD. Using beta-carotene as substrate, the enzyme activity was detected spectrophotometrically at a wavelength of 505 nm. The time constant of the reaction was 8.2 min, the Michaelis constant (K(m)) was 11.0 micromol x L(-1), and the maximum velocity (v(max)) was 0.083 micromol x L(-1) x min(-1) x mg(protein)(-1). The optimum temperature was above 50 degrees C.     

Study and characterization of polyphenol oxidase from eggplant (Solanum melongena L.)

In this study the catecholase and cresolase activities of eggplant polyphenol oxidase (PPO) were investigated. Enzyme activity was determined by measuring the increase in absorbance using catechol as substrate and 3-methyl-2-benzothiazolinone hydrazone (MBTH) as coupled reagent. The effects of substrate specificity, heat inactivation, temperature, pH, and inhibitors were investigated to understand the enzymatic alteration of ready-to-eat preparations. Browning of vegetables was determined through a colorimeter. Decrease of lightness (L*) and increase of color difference values (ΔE*) were correlated with tissue browning. Antibrowning agents were tested on PPO under the same conditions. The enzyme activity was strongly inhibited by 0.4 M citric acid. Under natural pH conditions, the enzyme was also inhibited by tartaric acid and acetic acid. All of the results were used to understand the best conditions for food transformation (ready-to-eat and grilled eggplant slices).     

Evidence for a tetrameric form of iceberg lettuce (Lactuca sativa L.) polyphenol oxidase: purification and characterization

Polyphenol oxidase from iceberg lettuce (Lactuca sativa L.) chloroplasts was released from the thylakoid-membrane by sonication, and it was extensively purified to homogeneity as judged by SDS-PAGE. Purification was achieved by ammonium sulfate fractionation, gel-filtration chromatography, and ion-exchange chromatography. Two molecular forms were separated by gel-filtration chromatography with apparent molecular masses of 188 and 49 kDa. Both forms were characterized by sedimentation analysis with S(20,W) values of 10.2 and 4.1 S, respectively. For the high-molecular-weight form purified to homogeneity, denaturing SDS-PAGE indicated a molecular mass of 60 kDa. Thus, from these data we suggest that lettuce polyphenol oxidase is a tetramer of identical subunits.     

Change in particle size of pectin reacted with pectinesterase isozymes from pea (Pisum sativum L.) sprout

Four pectinesterase (PE) isozymes were isolated by CM-Sepharose CL-6B chromatography from etiolated pea (Pisum sativum L.) sprouts and then reacted with citrus pectin (degree of esterification = 68%, 30-100 kDa) to observe the change in pectin particle size using a laser particle size analyzer. After incubation of a pectin-PE mixture (pH 6.5) at 30 degrees C for 4 h, PE 1 was observed to catalyze the transacylation reaction most remarkably, increasing the particle size from approximately 50-70 to approximately 250-350 nm, followed by PE 3, PE 2, and PE 4.     

Renodulation and characterization of Rhizobium isolates from cicer milkvetch (Astragalus cicer L.)

In 1993 and 1994, 12 bacterial isolates were isolated from root nodules of cicer milkvetch (Astragalus cicer). In the tests for nodulation of A. cicer by these bacterial isolates, five were found to form hypertrophic structures, while only two formed true nodules. These true nodules were formed in a sterilized soil system. This system might be able to act as a DNA donor to provide residual DNA to other microbes in the soil. The rhizobial isolates were thought to have lost genetic material crucial to nodulation during the isolation process. This hypothesis was supported by an experiment in which isolate B2 was able to nodulate A. cicer in vermiculite culture after being mixed with heat-killed rhizobia, Rhizobium leguminosarum bv. trifolii and R. loti. The nodulation would not occur in vermiculite culture system without the heat-killed rhizobia. Based on the biochemical data, the B2 and 9462L, which formed true nodules with A. cicer, were closely related. The rhizobia type cultures that nodulate A. cicer include Bradyrhizobium japonicum, Rhizobium leguminosarum bv. trifolii, R. leguminosarum bv. viceae, and R. loti. All of these rhizobia were from different cross-inoculation groups. The B2 and 9462L isolates could only nodulate Medicago sativa, Phaseolus vulgaris, and Melilotus officinalis, but not these species within the genus from which they were isolated: Astragalus. The traditional cross-inoculation group concept obviously does not fit well in the classification of rhizobia associated with Astragalus. The rhizobia isolated from A. cicer can be quite different, and the rhizobia able to renodulate A. cicer also quite diverse. Received: 27 June 1996