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).     

Purification and characterization of a polyphenol oxidase from the seeds of field bean (Dolichos lablab)

The polyphenol oxidase from field bean (Dolichos lablab) seeds has been purified to apparent homogeneity by a combination of ammonium sulfate precipitation, DEAE-Sephacel chromatography, phenyl agarose chromatography, and Sephadex G-200 gel filtration. The purified enzyme has a molecular weight of 120 +/- 3 kDa and is a tetramer of 30 +/- 1.5 kDa. Native polyacrylamide gel electrophoresis of the purified enzyme revealed the presence of a single isoform with an observed pH optimum of 4.0. 4-Methyl catechol is the best substrate, followed by catechol, and L-3,4-dihydroxyphenylalanine, all of which exhibited a phenomenon of inhibition by excess substrate. No activity was detected toward chlorogenic acid, catechin, caffeic acid, gallic acid, and monophenols. Tropolone, both a substrate analogue and metal chelator, proved to be the most effective competitive inhibitor with an apparent K(i) of 5.8 x 10(-)(7) M. Ascorbic acid, metabisulfite, and cysteine were also competitive inhibitors.     

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.     

Partial purification of latent persimmon fruit polyphenol oxidase

Persimmon fruit polyphenol oxidase (PPO) was partially purified using a combination of phase partitioning with Triton X-114 and ammonium sulfate fractionation between 50 and 75%. The enzyme, which showed both monophenolase and diphenolase activities, was partially purified in a latent form and could be optimally activated by the presence of 1 mM sodium dodecyl sulfate (SDS) with an optimum pH of 5.5. In the absence of SDS, the enzyme showed maximum activity at acid pH. SDS-PAGE showed the presence of a single band when L-DOPA was used as substrate. The apparent kinetic parameters of the latent enzyme were determined at pH 5.5, the V(m) value being 15 times higher in the presence of SDS than in its absence, whereas the K(M) was the same in both cases, with a value of 0.68 mM. The effect of several inhibitors was studied, tropolone being the most active with a K(i) value of 0.45 microM. In addition, the effect of cyclodextrins (CDs) was studied, and the complexation constant (K(c)) between 4-tert-butylcatechol (TBC) and CDs was calculated using an enzymatic method. The value obtained for K(c) was 15580 M(-1).     

Purification and characterization of polyphenol oxidase from banana (Musa sapientum L.) pulp

Polyphenol oxidase (EC 1.10.3.1, PPO) in the pulp of banana (Musa sapientum L.) was purified to 636-fold with a recovery of 3.0%, using dopamine as substrate. The purified enzyme exhibited a clear single band on polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS)-PAGE. The molecular weight of the enzyme was estimated to be about 41000 and 42000 by gel filtration and SDS-PAGE, respectively. The enzyme quickly oxidized dopamine, and its K(m) value for dopamine was 2.8 mM. The optimum pH was at 6.5, and the enzyme activity was stable in the range of pH 5-11 at 5 degrees C for 48 h. The enzyme had an optimum temperature of 30 degrees C and was stable even after a heat treatment at 70 degrees C for 30 min. The enzyme activity was completely inhibited by L-ascorbic acid, cysteine, sodium diethyldithiocarbamate, and potassium cyanide. Under a low buffer capacity, the enzyme was also strongly inhibited by citric acid and acetic acid at 10 mM.     

Catalytic properties of alcohol acyltransferase in different strawberry species and cultivars

The substrate specificity of alcohol acyltransferase (AAT) enzymes from different strawberry varieties was studied. Proteins with AAT activity from fruits of Fragaria x ananassa Duch. cv. Oso Grande were purified to apparent homogeneity and used for kinetic studies with different straight-chain alcohols and acyl-CoAs. K(m) values obtained for Oso Grande enzyme with six different alcohols, using acetyl-CoA as cosubstrate, decreased with increasing length of the alcohol chain. In similar experiments the increase in the acyl-CoA carbon chain was also found to be correlated with a higher substrate specificity. Heptanol (K(m) = 0.73 mM) and hexanoyl-CoA (K(m) = 0.41 mM) were the best substrates for Oso Grande AAT. Comparative catalytic studies were carried out with AAT partially purified extracts from the wild type Fragaria vesca and five commercial strawberry varieties: Tudnew, Carisma, Camarosa, Sweet Charlie, and Eris. The specificities of these enzymes toward five selected alcohols and acyl-CoAs reflected interesting cultivar differences.     

Aroma biosynthesis in strawberry: s-adenosylmethionine:furaneol o-methyltransferase activity in ripening fruits

Among the most important volatile compounds in the aroma of strawberries are 2,5-dimethyl-4-hydroxy-3(2H)-furanone (Furaneol) and its methoxy derivative (methoxyfuraneol, mesifuran). Three strawberry varieties, Malach, Tamar, and Yael, were assessed for total volatiles, Furaneol, and methoxyfuraneol. The content of these compounds sharply increased during fruit ripening, with maximum values at the ripe stage. An enzymatic activity that transfers a methyl group from S-adenosylmethionine (SAM) to Furaneol sharply increases during ripening of strawberry fruits. The in vitro generated methoxyfuraneol was identified by radio-TLC and GC-MS. The partially purified enzyme had a native molecular mass of approximately 80 kDa, with optimum activity at pH 8.5 and 37 degrees C. A high apparent K(m) of 5 mM was calculated for Furaneol, whereas this enzyme preparation apparently accepted as substrates other o-dihydroxyphenol derivatives (such as catechol, caffeic acid, and protocatechuic aldehyde) with much higher affinities (K(m) approximately 105, 130, and 20 microM, respectively). A K(m) for SAM was found to be approximately 5 microM, regardless of the acceptor used. Substrates that contained a phenolic group with only one OH group, such as p-coumaric and trans-ferulic acid, as well as trans-anol and coniferyl alcohol, were apparently not accepted by this activity. It is suggested that Furaneol methylation is mediated by an O-methyltransferase activity and that this activity increases during fruit ripening.     

Purification and characterization of a novel β-1,3-1,4-glucanase (lichenase) from thermophilic Rhizomucor miehei with high specific activity and its gene sequence

Production, purification, and characterization of a novel β-1,3-1,4-glucanase (lichenase) from thermophilic Rhizomucor miehei CAU432 were investigated. High-level extracellular β-1,3-1,4-glucanase production of 6230 U/mL was obtained when oat flour (3%, w/v) was used as a carbon source at 50 °C. The crude enzyme was purified to homogeneity with a specific activity of 28818 U/mg. The molecular weight of purified enzyme was estimated to be 35.4 kDa and 33.7 kDa by SDS-PAGE and gel filtration, respectively. The optimal pH and temperature of the enzyme were pH 5.5 and 60 °C, respectively. The K(m) values of purified β-1,3-1,4-glucanase for barley β-glucan and lichenan were 2.0 mM and 1.4 mM, respectively. Furthermore, the gene (RmLic16A) encoding the β-1,3-1,4-glucanase was cloned and its deduced amino acid sequence showed the highest identity (50%) to characterized β-1,3-1,4-glucanase from Paecilomyces thermophila. The high-level production and biochemical properties of the enzyme enable its potential industrial applications.     

Purification and characterization of ferulic acid esterase from malted finger millet (Eleusine coracana, Indaf-15)

Ferulic acid esterase (EC 3.1.1.73) cleaves the feruloyl groups substituted at the 5'-OH group of arabinosyl residues of arabinoxylans and is known to modulate their functional properties. In this study, ferulic acid esterase from 96 h finger millet malt was purified to apparent homogeneity by three-step purification with a recovery of 3% and a fold purification of 22. The substrate p-nitrophenylferulate (PNPF) was synthesized and used to assay this enzyme spectrophotometrically. The products liberated from ragi and wheat water-soluble polysaccharides by the action of purified ragi ferulic acid esterase were identified by ESI-MS. The pH and temperature optima of the enzyme were found to be 6.0 and 45 degrees C, respectively. The pH and temperature stabilities of the enzyme were found to be in the range of 5.5-9.0 and 30 degrees C, respectively. The activation energy of the enzymatic reaction was found to be 4.08 kJ mol(-1). The apparent K m and V max of the purified ferulic acid esterase for PNPF were 0.053 microM and 0.085 unit mL(-1), respectively. The enzyme is a monomer with a molecular mass of 16.5 kDa. Metal ions such as Ni(2+), Zn(2+), Co(2+), and Cu(2+) and oxalic and citric acids enhanced the enzyme activity. The enzyme was completely inhibited by Fe(3+). Group specific reagents such as p-chloromercuric benzoate and iodoacetamide inhibited the enzyme, indicating the possible presence of cysteine residues in the active site pocket.     

Purification and characterization of a carboxypeptidase from squid hepatopancreas (Illex illecebrosus)

The hepatopancreas of squid (Illex illecebrosus) extract contains a wide range of carboxypeptidase (CP) activities based on hydrolysis of N-CBZ-dipeptide substrates. SDS-PAGE zymograms with N-CBZ-Phe-Leu substrate revealed three activity zones (CP-I, 23 kDa; CP-II, 29 kDa; CP-III, 42 kDa). CP-I was purified 225-fold with 86.20% recovery based on N-CBZ-Ala-Phe activity by chromatography on DEAE-cellulose, gel filtration, and chromatofocusing. The purified enzyme had broad specificity toward N-CBZ-dipeptides; however, it preferred substrates with a hydrophobic amino acid at the C terminus. CP-I had greatest activity with N-CBZ-Ala-Phe (specific activity = 7104 units/mg of protein, K(m) = 0.40 mM, and physiological efficiency = 22863). CP-I had a pI of 3.4 and is a metalloprotease that is activated by Co(2+) and partially inhibited by Pefabloc, a serine protease inhibitor. With N-CBZ-Ala-Phe and Gly-Phe, it had optimum activity at pH 8 and 70 degrees C. The amino acid composition of squid CP-I is similar to that of CP A from other species.     

Biochemical characterization of cystine lyase from broccoli (Brassica oleracea var. italica)

Cystine lyase is the enzyme responsible for off-aroma deterioration in fresh unblanched broccoli. In this research, cystine lyase purification from broccoli has been optimized. Only one protein peak with cystine lyase activity was found during purification. Broccoli cystine lyase was purified 100-fold to homogeneity. L-Cystine, L-cysteine-S-sulfate, L-djenkolic acid, and some S-alkyl-L-cysteines and their sulfoxides are substrates, but the enzyme had negligible activity with L-cystathionine. A K(m) value of 81.2 microM was found for L-cystine. Inhibition and K(i) determinations indicated that L-cysteine is a linear noncompetitive inhibitor with a K(i) of 5 mM and DL-homocysteine is a competitive inhibitor with a K(i) of 1.5 mM. The molecular weight of cystine lyase was determined to be 100 kDa by three methods, with two subunits of 48 kDa each and a carbohydrate content of 3%. Further characterization included cofactor quantification, the effects of temperature and pH on activity and stability, and amino acid composition.     

Purification and characterization of a stable cysteine protease ervatamin B, with two disulfide bridges, from the latex of Ervatamia coronaria

Latex of the medicinal plant Ervatamia coronaria was found to contain at least three cysteine proteases with high proteolytic activity, called ervatamins. One of these proteases, named ervatamin B, has been purified to homogeneity using ion-exchange chromatography and crystallization. The molecular mass of the enzyme was estimated to be 26 000 Da by SDS-PAGE and gel filtration. The extinction coefficient (epsilon(1%)(280 nm)) of the enzyme was 20.5 with 7 tryptophan and 10 tyrosine residues per molecule. The enzyme hydrolyzed denatured natural substrates such as casein, azoalbumin, and azocasein with a high specific activity. In addition, it showed amidolytic activity toward N-succinyl-alanine-alanine-alanine-p-nitroanilide with an apparent K(m) and K(cat) of 6.6 +/- 0.5 mM and 1.87 x 10(2) s(-)(1), respectively. The pH optima was 6.0-6.5 with azocasein as substrate and 7.0-7.5 with azoalbumin as substrate. The temperature optimum was around 50-55 degrees C. The enzyme was basic with an isoelectric point of 9.35 and had no carbohydrate content. Both the proteolytic and amidolytic activity of the enzyme was strongly inhibited by thiol-specific inhibitors. Interestingly, the enzyme had only two disulfide bridges versus three as in most plant cysteine proteases of the papain superfamily. The enzyme was relatively stable toward pH, denaturants, temperature, and organic solvents. Polyclonal antibodies raised against the pure enzyme gave a single precipitin line in Ouchterlony's double immunodiffusion and typical color in ELISA. Other related proteases do not cross-react with the antisera to ervatamin B showing that the enzyme is immunologically distinct. The N-terminal sequence showed conserved amino acid residues and considerable similarity to typical plant cysteine proteases.     

Beta-glucosidase from Chalara paradoxa CH32: purification and properties

The hyphomycete Chalara paradoxa CH32 produced an extracellular beta-glucosidase during the trophophase. The enzyme was purified to homogeneity by ion-exchange and size-exclusion chromatography. The purified enzyme had an estimated molecular mass of 170 kDa by size-exclusion chromatography and 167 kDa by SDS-PAGE. The enzyme had maximum activity at pH 4.0-5.0 and 45 degrees C. The enzyme was inactivated at 60 degrees C. At room temperature, it was unstable at acidic pH, but it was stable to alkaline pH. The purified enzyme was inhibited markedly by Hg(2+) and Ag(2+) and also to some extent by the detergents SDS, Tween 80, and Triton X-100 at 0.1%. Enzyme activity increased by 3-fold in the presence of 20% ethanol and to a lesser extent by other organic solvents. Purified beta-glucosidase was active against cellobiose and p-nitrophenyl-beta-D-glucopyranoside but did not hydrolyze lactose, maltose, sucrose, cellulosic substrates, or galactopyranoside, mannopyranoside, or xyloside derivatives of p-nitrophenol. The V(max) of the enzyme for p-NPG (K(m) = 0.52 mM) and cellobiose (K(m) = 0.58 mM) were 294 and 288.7 units/mg, respectively. Hydrolysis of pNPG was inhibited competitively by glucose (K(i) = 11.02 mM). Release of reducing sugars from carboxymethylcellulose by a purified endoglucanase produced by the same organism increased markedly in the presence of beta-glucosidase.     

Characterization of a heterodimeric GH2 β-galactosidase from Lactobacillus sakei Lb790 and formation of prebiotic galacto-oligosaccharides

The lacLM genes from Lactobacillus sakei Lb790, encoding a heterodimeric β-galactosidase that belongs to glycoside hydrolase family GH2, were cloned and heterologously expressed in Escherichia coli . Subsequently, the recombinant β-galactosidase LacLM was purified to apparent homogeneity and characterized. The enzyme is a β-galactosidase with narrow substrate specificity because o-nitrophenyl-β-D-galactopyranoside (oNPG) was efficiently hydrolyzed, whereas various structurally related oNP analogues were not. The K(m) and k(cat) values for oNPG and lactose were 0.6 mM and 180 s(-1) and 20 mM and 43 s(-1), respectively. The enzyme is inhibited competitively by its two end-products D-galactose and D-glucose (K(i) values of 180 and 475 mM, respectively). As judged by the ratio of the inhibition constant to the Michaelis constant, K(i)/K(m), this inhibition is only very moderate and much less pronounced than for other microbial β-galactosidases. β-Galactosidase from L. sakei possesses high transgalactosylation activity and was used for the synthesis of galacto-oligosaccharides (GalOS), employing lactose at a concentration of 215 g/L. The maximum GalOS yield was 41% (w/w) of total sugars at 77% lactose conversion and contained mainly non-lactose disaccharides, trisaccharides, and tetrasaccharides with approximately 38, 57, and 5% of total GalOS formed, respectively. The enzyme showed a strong preference for the formation of β-(1→6)-linked transgalactosylation products, whereas β-(1→3)-linked compounds were formed to a lesser extent and β-(1→4)-linked reaction products could not be detected.     

Isolation, purification, and characterization of a cold-active lipase from Aspergillus nidulans

Aspergillus nidulans WG312 strain secreted lipase activity when cultured in liquid media with olive oil as carbon source. Highest lipase productivity was found when the mycelium was grown at 30 degrees C in a rich medium. The new enzyme was purified to homogeneity from the extracellular culture of A. nidulans by phenyl-Sepharose chromatography and affinity binding on linolenic acid-agarose. The lipase was monomeric with an apparent M(r) of 29 kDa and a pI of 4.85 and showed no glycosylation. Kinetic of enzyme activity versus substrate concentration showed a typical lipase behavior, with K(M) and K(cat) values of 0.28 mM and 494 s(-)(1) and 0.30 mM and 320 s(-)(1) for the isotropic solution and for the turbid emulsion, respectively. All glycerides assayed were hydrolyzed efficiently by the enzyme, but this showed preference toward esters of short- and middle-chain fatty acids. The optimum temperature and pH for the lipolytic activity were 40 degrees C and 6.5, with high activity in the range 0-20 degrees C and reduced thermal stability.     

Purification and characterization of vanilla bean (Vanilla planifolia Andrews) beta-D-glucosidase

Vanilla bean beta-D-glucosidase was purified to apparent homogeneity by successive anion exchange, hydrophobic interaction, and size-exclusion chromatography. The enzyme is a tetramer (201 kDa) made up of four identical subunits (50 kDa). The optimum pH was 6.5, and the optimum temperature was 40 degrees C at pH 7.0. K(m) values for p-nitrophenyl-beta-D-glucopyranoside and glucovanillin were 1.1 and 20.0 mM, respectively; V(max) values were 4.5 and 5.0 microkat.mg(-1). The beta-D-glucosidase was competitively inhibited by glucono-delta-lactone and 1-deoxynojirimycin, with respective K(i) values of 670 and 152 microM, and not inhibited by 2 M glucose. The beta-D-glucosidase was not inhibited by N-ethylmaleimide and DTNB and fully inhibited by 1.5-2 M 2-mercaptoethanol and 1,4-dithiothreitol. The enzyme showed decreasing activity on p-nitrophenyl-beta-D-fucopyranoside, p-nitrophenyl-beta-D-glucopyranoside, p-nitrophenyl-beta-D-galactopyranoside, and p-nitrophenyl-beta-D-xylopyranoside. The enzyme was also active on prunasin, esculin, and salicin and inactive on cellobiose, gentiobiose, amygdalin, phloridzin, indoxyl-beta-D-glucopyranoside, and quercetin-3-beta-D-glucopyranoside.     

Purification and characterization of polyphenol oxidase from garland chrysanthemum (Chrysanthemum coronarium L.)

Polyphenol oxidase (PPO) of garland chrysanthemum (Chrysanthemum coronarium L.) was purified approximately 32-fold with a recovery rate of 16% by ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography, and gel filtration. The purified enzyme appeared as a single band on PAGE and SDS-PAGE. The molecular weight of the enzyme was estimated to be about 47000 and 45000 by gel filtration and SDS-PAGE, respectively. The purified enzyme quickly oxidized chlorogenic acid and (-)-epicatechin. The K(m) value (Michaelis constant) of the enzyme was 2.0 mM for chlorogenic acid (pH 4.0, 30 degrees C) and 10.0 mM for (-)-epicatechin (pH 8.0, 40 degrees C). The optimum pH was 4.0 for chlorogenic acid oxidase (ChO) and 8.0 for (-)-epicatechin oxidase (EpO). In the pH range from 5 to 11, their activities were quite stable at 5 degrees C for 22 h. The optimum temperatures of ChO and EpO activities were 30 and 40 degrees C, respectively. Both activities were stable at up to 50 degrees C after heat treatment for 30 min. The purified enzyme was strongly inhibited by l-ascorbic acid and l-cysteine at 1 mM.     

Purification and characterization of a beta-glucosidase from Citrus sinensis var. Valencia fruit tissue

A preliminary survey demonstrated activity for alpha-D-glucosidase, alpha-D-mannosidase, alpha-L-arabinosidase, beta-D-glucosidase, beta-D-xylosidase, and beta-D-galactosidase in orange fruit flavedo and albedo tissue. alpha-L-Rhamnosidase was not detected. Subsequently, a beta-glucosidase was purified from mature fruit rag tissue (composed of intersegmental septa, squeezed juice sacs, and fruit core tissue) of Citrus sinensis var. Valencia. The beta-glucosidase exhibited low levels of activity against p-nitrophenyl-beta-D-fucopyranoside (13.5%) and p-nitrophenyl-alpha-D-glucopyranoside (7.0%), compared to its activity against p-nitrophenyl-beta-D-glucopyranoside (pNPG, 100%). The enzyme was purified by a combination of ion exchange (anion and cation) and gel filtration (Superdex and Toyopearl HW-55S) chromatography. It has an apparent molecular mass of 64 kDa by denaturing electrophoresis or 55 kDa by gel filtration chromatography (BioGel P-100). Hydrolysis of pNPG demonstrated a pH optimum between 4.5 and 5.5. At pH 5.0 the temperature optimum was 40 degrees C. At pH 5.0 and 40 degrees C the K(m) for pNPG was 0.1146 mM and it had a V(max) of 5.2792 nkatal x mg(-1) protein (katal = 0.06 International Units = the amount of enzyme that produces, under standard conditions, one micromol of product per min). Of the substrates tested, the enzyme was most active against the disaccharide cellobiose (1-->4), but was not active against p-nitrophenyl-beta-D-cellobioside. High levels of activity also were observed with the disaccharides laminaribiose (1-->3), gentiobiose (1-->6), and sophorose (1-->2). Activity greater than that observed with pNPG was obtained with the flavonoids hesperetin-7-glucoside and prunin (naringenin-7-glucoside), salicin, mandelonitrile-beta-D-glucoside (a cyanogenic substrate), and sinigrin (a glucosinolate). The enzyme was not active against amygdalin, coniferin, or limonin glucoside.     

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.     

Chemical heterogeneity of humic substances: characterization of size fractions obtained by hollow-fibre ultrafiltration

To investigate the chemical heterogeneity of humic substances in relation to molecular size, fulvic and humic acids were extracted and purified from the surface horizon of a Humic Gleysol in northern Switzerland. A fractionation scheme using hollow‐fibre ultrafiltration cartridges was developed and used to obtain four size fractions of the humic acid with nominal molecular weight ranges > 300 kDa, 100–300 kDa, 30–100 kDa, and 10–30 kDa. The fulvic acid and all humic acid fractions were characterized by size exclusion chromatography, elemental analysis (C, H, N, S), as well as spectroscopic techniques including UV‐VIS, CP‐MAS ¹³C‐NMR, FT‐IR, and fluorescence spectroscopy. Clear chemical differences between the humic acid size fractions were observed. Smaller size fractions of the soil humic acid contained more chargeable functional groups and a larger percentage of aromatic carbon than the larger size fractions. Conversely, the percentage of aliphatic carbon increased with increasing apparent molecular weight. The chemical composition of the smallest humic acid fraction differed clearly from the fulvic acid fraction, despite similar apparent molecular size and carboxyl carbon content. Small humic acids contained much more aromatic carbon and less aliphatic carbon than the fulvic acid fraction. Apparently, humic size fractions differ in their chemical composition, which can have important implications for their environmental behaviour.