Purification and properties of 4-methyleneglutaminase from the leaves of peanut (Arachis hypogaea)

The enzyme 4-methyleneglutaminase has been purified from Arachis hypogaea leaves. This enzyme also catalysed the deamidation of glutamine at 20% of the rate of 4-methyleneglutamine, exhibiting the same affinity for both substrates (K_m  20 mM), but was inactive with asparagine. The hydrolysis of 4-methyleneglutamine was subject to competitive inhibition by glutamine, glutamate-5-hydroxamate and phenol red and non-competitive inhibition by glutamate and 4-methyleneglutamate. The enzyme activity was insensitive to a variety of salts and carboxylic acids.     

Purification and partial biochemical characterization of polyphenol oxidase from mamey (Pouteria sapota)

While a long shelf life for fruit products is highly desired, enzymatic browning is the main cause of quality loss in fruits and is therefore a main problem for the food industry. In this study polyphenol oxidase (PPO), the main enzyme responsible for browning was isolated from mamey fruit (Pouteria sapota) and characterized biochemically. Two isoenzymes (PPO 1 and PPO 2) were obtained upon ammonium sulfate precipitation and hydrophobic and ion exchange chromatography; PPO 1 was purified up to 6.6-fold with 0.28% yield, while PPO 2 could not be characterized as enzyme activity was completely lost after 24 h of storage. PPO 1 molecular weight was estimated to be 16.1 and 18 kDa by gel filtration and SDS-PAGE, respectively, indicating that the native state of the PPO 1 is a monomer. The optimum pH for PPO 1 activity was 7. The PPO 1 was determined to be maximum thermally stable up to 35 °C. Kinetic constants for PPO 1 were K_m = 44 mM and K_m = 1.3 mM using catechol and pyrogallol as substrate, respectively. The best substrates for PPO 1 were pyrogallol, 4-methylcatechol and catechol, while ascorbic acid and sodium metabisulfite were the most effective inhibitors.     

Some kinetic properties of polyphenol oxidase obtained from dill (Anethum graveolens)

Polyphenol oxidase (PPO) was partially purified from dill by (NH₄)₂SO₄ precipitation followed by dialysis and gel filtration chromatography. Polyphenol oxidase activity was measured spectrophotometrically at 420 nm using catechol, dopamine and chlorogenic acid as substrates. Optimum pH, temperature, and ionic strength were determined with three substrates. The best substrate of dill PPO was found to be chlorogenic acid. Some kinetic properties of the enzyme such as V_max, K_M and V_max/K_M were determined for all three substrates. The effects of various inhibitors on the reaction catalysed by the enzyme were tested and I₅₀ values calculated. The most effective inhibitor was l-cysteine. Activation energies, E_a, were determined from the Arrhenius equation. In addition, activation enthalpy, ΔH_a, and Q₁₀ values of the enzyme were also calculated.     

o-Diphenol: Oxygen oxidoreductase from leaves of sugar cane

A non-particulate o-diphenol: O₂ oxidoreductase (phenolase) has been isolated from leaves of sugar cane. Gel filtration produced two fractions MW 32000 and 130000. The preferred substrate was chlorogenic acid. Other o-diphenols (caffeic acid, catechol, pyrogallol, dihydroxyphenylalanine) all of which were slowly oxidized when tested alone, increased the rates of O₂ consumption obtained with catalytic amounts of chlorogenic acid. Both enzyme fractions were inhibited by thiols; thioglycollate, which acted in a non-competitive manner, was most effective.     

Purification,characterization and partial amino acid sequences of a novel cephalosporin-C deacetylase from Bacillus subtilis

Cephalosporin-C deacetylase [EC 3.1.1.41] was purified electrophoretically to homogeneity from the newly isolated Bacillus subtilis SHS 0133 (FERM BP-2755). The enzyme was purified about 27-fold with a yield of 9 % and a specific activity of 187.4 U/mg protein. The native enzyme (molecular weight, 280,000) was composed of eight identical subunits with apparent molecular weights of 35,000. The cephalosporin-C deacetylase was stable up to 60°C for 30 min at pH 7.0. The enzyme exhibited Michaelis-Menten kinetics with the substrates cephalosporin C, 7-aminocephalosporanic acid (7-ACA) and p-nitrophenyl acetate; the K_m values were 24.0, 7.9 and 1.0 mM, respectively. One of the reaction products from 7-ACA, deacetyl-7-ACA, was a weak non-competitive inhibitor and other product, acetate, was a weak competitive inhibitor; the K_i values were 171 and 290 mM, respectively. However, these weak product inhibitors did not prevent the completion of the deacylation of 7-ACA. The pI value of the enzyme was determined to be 5.3 using isoelectric focusing. The observed data indicate that the enzyme is different from known cephalosporin-C deacetylases. In addition, amino acid sequencing of the N-terminus and Achromobacter proteinase I-digested peptides yielded no sequences with similarities to other known proteins by a computer search.     

Oxidation of catechol in plants. 3. Purification and properties of the diphenylene dioxide 2,3-quinone-forming enzyme system from Tecoma leaves

In attempting to determine the nature of the enzyme system mediating the conversion of catechol to diphenylenedioxide 2,3-quinone, in Tecoma leaves, further purification of the enzyme was undertaken. The crude enzyme from Tecoma leaves was processed further by protamine sulfate precipitation, positive adsorption on tricalcium phosphate gel, and elution and chromatography on DEAE-Sephadex. This procedure yielded a 120-fold purified enzyme which stoichiometrically converted catechol to diphenylenedioxide 2,3-quinone. The purity of the enzyme system was assessed by polyacrylamide gel electrophoresis. The approximate molecular weight of the enzyme was assessed as 200,000 by gel filtration on Sephadex G-150. The enzyme functioned optimally at pH 7.1 and at 35 °C. The K_m for catechol was determined as 4 × 10^?4m. The enzyme did not oxidize o-dihydric phenols other than catechol and it did not exhibit any activity toward monohydric and trihydric phenols and flavonoids. Copper-chelating agents did not inhibit the enzyme activity. Copper could not be detected in the purified enzyme preparations. The purified enzyme was not affected by extensive dialysis against copper-complexing agents. It did not show any peroxidase activity and it was not inhibited by catalase. Hydrogen peroxide formation could not be detected during the catalytic reaction. The enzymatic conversion of catechol to diphenylenedioxide 2,3-quinone by the purified Tecoma leaf enzyme was suppressed by such reducing agents as GSH and cysteamine. The purified enzyme was not sensitive to carbon monoxide. It was not inhibited by thiol inhibitors. The Tecoma leaf was found to be localized in the soluble fraction of the cell. Treatment of the purified enzyme with acid, alkali, and urea led to the progressive denaturation of the enzyme.     

Location and Catalytic Characteristics of a Multipotent Bacterial Polyphenol Oxidase

The melanogenic marine bacterium Marinomonas mediterranea contains a multipotent polyphenol oxidase (PPO) able to oxidize substrates characteristic for tyrosinase and laccase. Thus, this enzyme shows tyrosine hydroxylase activity and it catalyzes the oxidation of a wide variety of o-diphenol as well as o-methoxy-activated phenols. The study of its sensitivity to different inhibitors also revealed intermediate features between laccase and tyrosinase. It is similar to tyrosinases in its sensitivity to tropolone, but it resembles laccases in its resistance to cinnamic acid and phenylthiourea, and in its sensitivity to fluoride anion. This enzyme is mostly membrane-bound and can be solubilized either by non-ionic detergent or lipase treatments of the membrane. The expression of this enzymatic activity is growth-phase regulated, reaching a maximum in the stationary phase of bacterial growth, but l -tyrosine, Cu(II) ions, or 2,5-xylidine do not induce it. This enzyme can be separated from a second PPO form by gel permeation chromatography. The second PPO is located in the soluble fraction and shows a sodium dodecyl sulfate (SDS)-activated action on the characteristic substrates for tyrosinase, l -tyrosine, and l -dopa, but it does not show activity towards laccase-specific substrates. The involvement of the multipotent PPO in melanogenesis and its relationship with the SDS-activated form and with the alternative functions proposed for multicopper oxidases in other microorganisms are discussed.     

l-proline dehydrogenase of Triticum vulgare germ: Purification,properties and cofactor interactions

The enzyme catalysing the l-proline-dependent reduction of NAD⁺has been purified over 600-fold from wheat germ acetone powder extracts. l-Proline, 3,4 dehydro-dl-proline, thiazolidine-4-carboxylate were the only substrates utilized readily. The K_m for l-proline was 1·0 mM and for NAD⁺ 0·8 mM. The enzyme was highly specific for NAD⁺ with NADP⁺ and NADPH acting as effective competitive inhibitors with a K_i of 1·8 and 0·4 μM, respectively. All ribonucleoside triphosphates tested were good non-competitive inhibitors, in particular UTP. The purified enzyme could reduce pyrroline-5-carboxylate, either chemically synthesized or generated in a linked assay system from ornithine by a highly-purified ornithine transaminase. In the latter case both NADH and NADPH were utilized equally well as the reductant. With chemically synthesized dl-pyrroline-5-carboxy-late as the substrate. NADPH was used at only 25% the rate of NADH, and NADPH strongly inhibited the oxidation of NADH.     

槐尺蠖多酚氧化酶的纯化及酶学特征

经40%饱和度硫酸铵分级沉淀,Sephadex G-100凝胶过滤等步骤,将槐尺蠖Semiothisa cinerearia Bremer et Grey 多酚氧化酶纯化,纯化倍数为6.96倍。该酶对焦性没食子酸,邻苯二酚和L多巴的Km值分别为0.23 mmol/L, 0.48 mmol/L和0.49 mmol/L。多酚氧化酶在pH 7.0,37℃时活性最高,并在40℃以上条件下,随着保温时间的延长酶活力下降。用槲皮苷和硫脲作抑制剂对该酶活性的抑制结果表明,这两种抑制剂分别属于竞争性和非竞争性抑制剂。     

Characterization of wheat o-diphenolase isoenzyme

A highly purified isoenzyme of wheat o-diphenolase was characterized. The isoenzyme had a MW of ca 115 000, as determined by Sephadex G-100 gel filtration. The copper content was 0.20%, and the amino acid composition was determined. Two subunits (MWs ca 30 000 and 23 500) were detected by SDS gel electrophoresis. The K_m was found to be 5.1 mM for 4-methylcatechol and kinetic analysis showed that the isoenzyme exhibited substrate inhibition. The isoenzyme was characterized by its response to some inhibitors.     

Purification and properties of caffeic acid O-methyltransferase from alfalfa root nodules

An O-methyltransferase which catalyses the methylation of caffeic acid to ferulic acid using S-adenosyl-l-methionine as methyl donor has been isolated and purified ca 70-fold from root nodules of alfalfa. The enzyme also catalysed the methylation of 5-hydroxyferulic acid. Chromatography on 1,6-diaminohexane agarose (AH-Sepharose-4B) linked with S-adenosyl-l-homocysteine (SAH) gave 35% recovery of enzyme activity. The K_m values for caffeic acid and S-adenosyl-l-methionine were 58 and 4.1 μM, respectively. S-Adenosyl-l-homocysteine was a potent competitive inhibitor of S-adenosyl-l-methionine with a K_i of 0.44 μM. The MW of the enzyme was ca 103 000 determined by gel filtration chromatography.     

Purification and partial characterization of NAD aminohydrolase from Aspergillus oryzae NRRL447

Aspergillus oryzae aminohydrolase free acid phosphodiesterase catalyzes nicotinamide adenine dinucleotide to deamino-NAD and ammonia. The enzyme was purified to homogeneity by a combination of acetone precipitation, anion exchange chromatography and gel filtration chromatography. The enzyme was purified 230.5 fold. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band of MW 94 kDa. The enzyme displayed maximum activity at pH 5 and 40 °C with NAD as substrate. The enzyme activity appeared to be stable up to 40 °C. The enzyme activity was enhanced slightly by addition of Na⁺ and K⁺, whereas inhibited strongly by addition of Ag⁺, Mn²⁺, Hg²⁺ and Cu²⁺ to the reaction mixtures. The enzyme hydrolyzes several substrates, suggesting a probable non-specific nature. The enzyme catalyzes the hydrolytic cleavage of amino group of NAD, adenosine, AMP, CMP, GMP, adenosine, cytidine and cytosine to the corresponding nucleotides, nucleosides or bases and ammonia. The substrate concentration–activity relationship is the hyperbolic type and the apparent K_m and K_cat for the tested substrates were calculated.     

Properties of phenylalanine ammonia-lyase extracted from Cucumis sativus hypocotyls

Some of the in vitro properties of PAL from gherkin hypocotyls were investigated. No metal ion requirement for this enzyme could be demonstrated but there were indications that PAL was a sulphydryl enzyme. Kinetic analysis suggested that PAL exhibited negative homotropic cooperativity. Two K_m values were determined, these were K_m^H, 2·9 × 10^?4 M and K_m^L, 4·3 × 10^?5 M. Strong inhibition of the enzyme was exerted by d-phenylalanine, trans-cinnamic acid, o-coumaric acid, gallic acid, quercetin and kaempferol. Kinetic studies on the inhibition patterns of these compounds established that d-phenylalanine linearized the curvilinear kinetics, trans-cinnamic acid and o-coumaric acid acted as competitive inhibitors whilst gallic acid, quercetin and kaempferol acted as mixed inhibitors. Using a number of desensitization techniques PAL was partially desensitized to inhibition by the mixed inhibitors. These results led to the conclusion that PAL may have a regulatory role in phenol, coumarin and flavonoid biosynthesis.     

An inhibitor of phosphoinositol kinase from ungerminated mung bean seeds

A protein inhibitor of phosphoinositol kinase has been detected in the later stages of ripening of mung bean seeds. This has been isolated and purified from the ungerminated seeds. It migrated as a single protein band when subjected to polyacrylamide gel electrophoresis. The MW of the inhibitor is approx. 86 000. The phosphoinositol kinase inhibition has been found to be dependent on the protein concentration of the purified inhibitor. It seems that 1 molecule of the inhibitor is necessary to inhibit 1 molecule of enzyme. The nature of the inhibition has been found to be non-competitive, the K_i of which is around 1·47 × 10^?6 M. The enzyme inhibitor complex dissociates on gel electrophoresis without any loss of enzyme activity.     

Purification and some properties of a (1→4)-β-d-glucan glucohydrolase associated with the cellulase from the fungus Penicillium funiculosum

The (1→4)-β-d-glucan glucohydrolase from Penicillium funiculosum cellulase was purified to homogeneity by chromatography on DEAE-Sephadex and by iso-electric focusing. The purified component, which had a molecular weight of 65,000 and a pI of 4.65, showed activity on H₃PO₄-swollen cellulose, o-nitrophenyl β-d-glucopyranoside, cellobiose, cellotriose, cellotetraose, and cellopentaose, the K_m values being 172 mg/mL, and 0.77, 10.0, 0.44, 0.77, and 0.37 mm, respectively. d-Glucono-1,5-lactone was a powerful inhibitor of the action of the enzyme on o-nitrophenyl β-d-glucopyranoside (K_i 2.1 μm), cellobiose (K_i 1.95 μm), and cellotriose (K_i 7.9 μm) [cf.d-glucose (K_i 1756 μm)]. On the basis of a Dixon plot, the hydrolysis of o-nitrophenyl β-d-glucopyranoside appeared to be competitively inhibited by d-glucono-1,5-lactone. However, inhibition of hydrolysis by d-glucose was non-competitive, as was that for the gluconolactone-cellobiose and gluconolactone-cellotriose systems. Sophorose, laminaribiose, and gentiobiose were attacked at different rates, but the action on soluble O-(carboxymethyl)cellulose was minimal. The enzyme did not act in synergism with the endo-(1→4)-β-d-glucanase component to solubilise highly ordered cotton cellulose, a behaviour which contrasts with that of the other exo-(1→4)-β-d-glucanase found in the same cellulase, namely, the (1→4)-β-d-glucan cellobiohydrolase.     

Polyphenoloxidase from six mature grape varieties and their activities towards various phenols

Juices were prepared from three white and three red grape varieties harvested at full maturity and comparative studies on their oxygen-uptake, absorbance at 420 nm (degree of browning), polyphenoloxidase (EC 1.10.3.1; PPO) activity, and their phenol compositions were done. There was no correlation among the amounts of oxygen-uptake and oxidizable phenols in the juices and their degree of browning. However, there was similarity among the PPO from the six grape varieties in their general enzymatic properties and substrate specificity towards twenty-five phenols. A partially purified PPO fraction from Koshu juice, which did not contain free phenols, showed strong activity towards (+)-catechin, (−)-epicatechin, caffeic acid, catechol, pyrogallol, and protocatechuic acid (oxidizable phenols), but had no activity towards the other fifiteen phenols. The oxidizable substrates were not always the only limiting factor in the oxidation and browning of phenols by the PPO. Some unoxidizable phenols such as gallic acid, p-cresol, and tannic acid which were not substrates for PPO inhibited the oxidation of the oxidizable phenols except pyrogallol which was not inhibited by gallic acid. On the other hand, hydroquinone promoted the oxidation of the oxidizable phenols except protocatechuic acid. These showed that there were competitive reactions and synergism during the enzymatic oxidation of phenols.     

花色苷的酶降解

综述了降解花色苷的酶类及其降解机理的研究进展.降解花色苷的酶有花色苷酶、多酚氧化酶、过氧化物酶和果胶酶.花色苷酶和果胶酶均能水解花色苷糖苷键产生花色素和糖,花色素很不稳定,因吡喃烊环极易开环可自发转换成无色衍生物.花色苷不能直接作为PPO或POD的底物;PPO和POD氧化、降解花色苷须依赖具邻二酚结构的其他酚类的存在,...     

Characterization,purification, and temperature/pressure stability of polyphenol oxidase extracted from plums (Prunus domestica)

In this study, polyphenol oxidase (PPO) was extracted from Prunus domestica and partially purified by ammonium sulfate precipitation, hydrophobic interaction chromatography, and ion exchange chromatography. The final purification step revealed a 32.81-fold purification, and the molecular mass was estimated to be 65 kDa by SDS-PAGE. The purified PPO showed enzymatic activity mainly toward five substrates, namely catechol, catechin, 4-methyl catechol, chlorogenic acid, and L-3,4-dihydroxyphenylalanine, whereas it showed no activity toward caffeic acid, ferulic acid, p-coumaric acid, p-cresol, and l-tyrosine. The optimum pH and temperature values were 6.0 and 25 °C, respectively. The enzyme showed high stability in the pH range of 5.0–7.0 and in the temperature range of 25–65 °C. The most effective inhibitors of this enzyme were found to be ascorbic acid and l-cysteine. The thermal inactivation followed a first-order kinetic model, with activation energy of E_a 150.46 ± 1.29 kJ/mol. PPO extracted from plum showed stability at high pressure, with enzyme activation at 500 MPa.     

Purification and properties of NADP-dependent non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from the green alga Chlamydomonas reinhardtii

NADP-dependent non-phosphorylating D-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.9), previously described in higher plants, has been now found to be present in eukaryotic green algae, but in neither cyanobacteria nor non-photosynthetic microorganisms. The enzyme from the unicellular green alga Chlamydomonas reinhardtii, strain 6145c, has been purified to apparent electrophoretic homogeneity. The non-phosphorylating enzyme was effectively separated from the NADP-dependent phosphorylating D-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) dye-ligand chromatography on Reactive Red-120 agarose. The purified enzyme exhibited an optimum pH in the 8.5–9.0 range and a specific activity of approx. 8 μmol·(mg protein)⁻¹·min⁻¹. The native protein was characterized as a homotetramer with a molecular weight of 190 000, a Stokes radius of 5.2 mn, and an isoelectric point of 6.9. From kinetic studies, K_m-values of 9.8 and 51 μM were calculated for NADP and D-glyceraldehyde 3-phosphate, respectively, an absolute specificity for both substrates being observed. L-Glyceraldehyde 3-phosphate was a potent non-competitive inhibior (K_i, 48 μM). The reaction products NADPH and D-3-phosphoglycerate inhibited enzyme activity in a competitive manner with respect to NADP (K_i, 78 μM) and D-glyceraldehyde 3-phosphate (K_i, 1.2 mM), respectively. Thermal inactivation occurred above 45°C and was effectively prevented by either substrate. The presence of essential vicinal thiol groups is suggested by the inactivation produced by diamide, with D-glyceraldehyde 3-phosphate, but not NADP, behaving as a protective agent. The enzyme's possible physiological role in photosynthetic metabolism is discussed briefly.     

High polyphenol oxidase activity and low titratable acidity in browning bamboo tissue culture

Summary Tissue browning that frequently results in the early death of bamboo shoots in vitro correlated directly with polyphenol oxidase (PPO, EC 1.10.3.1) activity and inversely with titratable acidity. It was unrelated to the level of endogenous phenols. During the course of culture, timing of PPO activity paralleled that of explant browning. Browning was highest among shoots cultured in a medium of pH 8, which was consistent with the pH optinum of the bamboo enzyme. The pH optimum was first determined with the crude enzyme, then verified with two purified isozymes. Stability of the bamboo PPO was also highest at pH 10. PPO activities of the severely browning Dendrocalamus latiflorus, the moderately browning Phyllostachys nigra, and the relatively non-browning Bambusa oldhamii were inhibited strongly by ascorbic acid, cysteine, sodium diethyldithiocarbamate, and sodium sulfite. But characterization of bamboo PPO according to enzyme inhibitors was not possible because enzyme extracts of the three species gave varied responses to the traditional substances. Nutrient medium addenda of some PPO inhibitors, namely ascorbic acid, cysteine, kojic acid, and thiourea, mainly enhanced browning. However, ferulic acid at 3 mM and lower concentrations reduced the number of brown shoots per culture, although not the percentage of cultures that browned. Polyvinylpyrrolidone failed completely to suppress browning. The two purified isozymes showed different temperature optima for PPO activity: 60°C and 65°C. The purified isozymes displayed a substrate preference for dopamine, or a cathecol oxidase characteristics.