Some biochemical properties of polyphenol oxidase from celery

Polyphenol oxidase (PPO, EC 1.14.18.1) was extracted from celery roots (Apium graveolens L.) with 0.1 M phosphate buffer, pH 7.0. The PPO was partially purified by (NH4)2SO4 and dialysis. Substrate specificity experiments were carried out with catechol, pyrogallol, L-DOPA, p-cresol, resorcinol, and tyrosine. The Km for pyrogallol, catechol, and L-DOPA were 4.5, 8.3, and 6.2mM, respectively, at 25 degrees C. Data for Vmax/Km values, which represent catalytic efficiency, show that pyrogallol has the highest value. The optimum pH and temperature were determined with catechol, pyrogallol, and L-DOPA. Optimum pH was 7.0 for catechol and L-DOPA, and 7.5 for pyrogallol. Optimum temperatures for maximum PPO activity were 25 degrees C for pyrogallol, 40 degrees C for catechol, and 45 degrees C for L-DOPA. Heat inactivation studies showed a decrease in enzymatic activity at temperatures above 60 degrees C. The order of inhibitor effectiveness was: L-cysteine > ascorbic acid > glycine > resorcinol > NaCl.     

Enzymatic Characterization and Functional Groups of Polyphenol Oxidase from the Pupae of Blowfly (Sarcophaga bullata)

Polyphenol oxidase (EC 1.14.18.1) was purified from the pupae of blowfly (Sarcophaga bullata) by a procedure involving ammonium sulfate fractionation and chromatography on DEAE-cellulose and Sephadex G-100. Kinetic characteristics of the enzyme were determined using L-DOPA as substrate. The specific activity of the enzyme was 770 U/mg, and the Michaelis constant (Km) was 1.5 +/- 0.1 mM (pH 6.8, 30 degrees C). Activity was maximal at 40 degrees C, pH 6.5. Chemical modification experiments demonstrated that cysteine and tryptophan residues are essential and arginine residues are not essential to the enzyme function. The enzyme is inhibited by quercetin with an IC50 of 0.20 +/- 0.06 mM. The inhibition is of competitive type, and the inhibition constant was determined to be 88 micro M.     

Electron acquisition system constructed from an NAD-independent D-lactate dehydrogenase and cytochrome c2 in Rhodopseudomonas palustris No. 7

The activities of NAD-independent D- and L-lactate dehydrogenases (D-LDH, L-LDH) were detected in Rhodopseudomonas palustris No. 7 grown photoanaerobically on lactate. One of these enzymes, D-LDH, was purified as an electrophoretically homogeneous protein (M(r), about 235,000; subunit M(r) about 57,000). The pI was 5.0. The optimum pH and temperature of the enzyme were pH 8.5 and 50 degrees C, respectively. The Km of the enzyme for D-lactate was 0.8 mM. The enzyme had narrow substrate specificity (D-lactate and DL-2-hydroxybutyrate). The enzymatic activity was competitively inhibited by oxalate (Ki, 0.12 mM). The enzyme contained a FAD cofactor. Cytochrome c(2) was purified from strain No. 7 as an electrophoretically homogeneous protein. Its pI was 9.4. Cytochrome c(2) was reduced by incubating with D-LDH and D-lactate.     

Performance evaluation of a silk protein-based matrix for the enzymatic conversion of tyrosine to L-DOPA

L-DOPA (3,4-dihydroxyphenyl-L-alanine), one of the most important intermediates in the melanin biosynthesis pathway, is used for the treatment of Parkinson's disease. With a view of developing a cheaper and more effective method for the bioconversion of tyrosine to L-DOPA, the potential and performance of a novel fibrous matrix prepared from Bombyx mori silk protein fibroin were evaluated for the immobilization of tyrosinase. Cross-linkage between fibroin and tyrosinase using glutaraldehyde was evident from Fourier transform infra red spectroscopy. Maximum product formation occurred when 1000 U enzyme was immobilized on 20 mg fibroin. The optimum conditions for maximal L-DOPA production using immobilized tyrosinase were 40 degrees C and pH 5.5, conditions that caused a 50% loss of free enzyme activity. Immobilized tyrosinase also showed to have a higher degree of stability during storage and it retained 80% of its original activity after repeated reuses. The efficiency of this immobilized tyrosinase system to produce L-DOPA was high, as evident from a high effectiveness factor, between 0.7 and 0.8, thereby making this method feasible for the large-scale production of L-DOPA.     

Purification and some properties of 1-aspartamido-beta-N-acetylglucosamine amidohydrolase from human liver.

Human liver 1-aspartamido-beta-N-acetylglucosamine amidohydrolase (aspartylglucosylaminase, EC 3.5.1.26) was purified 17 500-fold to apparent homogeneity as judged from polyacrylamide-gel disc electrophoresis. A pH optimum of 7.7-9.0 was found. The Km value was pH- and temperature-dependent. At 37 degrees C and pH 7.7, Km was 0.16 mM and it increased to 0.29 at pH 6.0 and 0.23 at pH 9.0. At 25 degrees C and pH 7.7, a Km value of 0.99 mM was obtained. When the substrate concentration was varied, apparent Michaelis-Menten kinetics were obtained. p-Hydroxymercuribenzoate, glutathione or cysteine had no effect on the enzyme activity; 5 mM-N-acetylcysteine inhibited about 47% of the total enzyme activity. Apart from Cu2+, other bivalent ions were virtually ineffective at 1 mM. The kinetic study differentiates this enzyme from aspartylglucosylaminase from other sources.     

Purification and characterization of hydroxypyruvate reductase from a serine-producing methylotroph, Hyphomicrobium methylovorum GM2

Hydroxypyruvate reductase of a serine-producing methylotroph, Hyphomicrobium methylovorum GM2, was purified to complete homogeneity, crystallized and characterized, the first time for an enzyme from a methylotroph. The enzyme was found to be a dimer composed of identical subunits (38 kDa), the molecular mass of the enzyme being about 70 kDa. The enzyme was stable against heating at 25 degrees C for 10 min at pH values between 5 and 9. Optimal activity was observed at pH 6.8 and around 45 degrees C. The enzyme catalyzed the reduction of hydroxypyruvate with the oxidation of only NADH. Other than hydroxypyruvate, only glyoxylate served as a substrate. The Km values were found to be 0.175 mM for hydroxypyruvate and 10.8 mM for glyoxylate. Taking advantage of the high substrate specificity of this enzyme, a means of enzymatic determination of hydroxypyruvate was established.     

Studies on methanol — oxidizing yeast

Oxidation of methanol, formaldehyde and formic acid was studied in cells and cell-free extract of the yeast Candida boidinii No. 11Bh. Methanol oxidase, an enzyme oxidizing methanol to formaldehyde, was formed inducibly after the addition of methanol to yeast cells. The oxidation of methanol by cell-free extract was dependent on the presence of oxygen and independent of any addition of nicotine-amide nucleotides. Temperature optimum for the oxidation of methanol to formaldehyde was 35 degrees C, pH optimum was 8.5. The Km for methanol was 0.8mM. The cell-free extract exhibited a broad substrate specificity towards primary alcohols (C1--C6). The activity of methanol oxidase was not inhibited by 1mM KCN, EDTA or monoiodoacetic acid. The strongest inhibitory action was exerted by p-chloromercuribenzoate. Both the cells and the cell-free extract contained catalase which participated in the oxidation of methanol to formaldehyde; the enzyme was constitutively formed by the yeast. The pH optimum for the degradation of H2O2 was in the same range as the optimum for methanol oxidation, viz. at 8.5. Catalase was more resistant to high pH than methanol oxidase. The cell-free extract contained also GSH-dependent NAD-formaldehyde dehydrogenase with Km = 0.29mM and NAD-formate dehydrogenase with Km = 55mM.     

Immobilization of tyrosinase on chitosan-clay composite beads

Tyrosinase was immobilized on glutaraldehyde crosslinked chitosan-clay composite beads and used for phenol removal. Immobilization yield, loading efficiency and activity of tyrosinase immobilized beads were found as 67%, 25% and 1400 U/g beads respectively. Optimum pH of the free and immobilized enzyme was found as pH 7.0. Optimum temperature of the free and immobilized enzyme was determined as 25-30 °C and 25 °C respectively. The kinetic parameters of free and immobilized tyrosinase were calculated using l-catechol as a substrate and K(m) value for free and immobilized tyrosinase were found as 0.93 mM and 1.7 mM respectively. After seven times of repeated tests, each over 150 min, the efficiency of phenol removal using same immobilized tyrosinase beads were decreased to 43%.     

A specific L-asparaginase from Thermus aquaticus

The L-asparaginase from an extreme thermophile, Thermus aquaticus strain T351, was highly substrate- and stereospecific, with no activity against glutamine or D-asparagine. It had a high Km of 8.6 mM. In these aspects it closely resembled the corresponding enzymes from thermophilic bacteria. The enzyme had a molecular weight of 80,000, an isoelectric point of 4.6, and a pH optimum of 9.5. It showed some substrate inhibition above 20 mM asparagine and was also inhibited by L-aspartic acid, D- and L-lysine (Ki of 5.2 and 1.25 mM, respectively), and D- and L-serine. The half-life of the enzyme at 85 degrees C was 40 min. The Arrhenius plot showed a change in slope at 55 degrees C.     

Phosphoenolpyruvate carboxylase from the roots of yellow lupin (Lupinus luteus)

A crude preparation of PEP carboxylase (EC 4.1.1.31) from the yellow lupin roots exhibits the pH optimum of activity within the range of 7.4-8.6 and the temperature optimum at 32 - 40 degrees C. Its Km for PEP is 0.1 mM, and Km for HCO3- is 0.7 mM. The affinity of the enzyme towards Mg2+ diminishes with the metal ion concentration. At the concentration of Mg2+ below 0.5 mM Km for Mg2+ is 0.07 mM and at the Mg2+ concentration over 1.5 mM it rises to 0.47 mM. The Hill coefficients are 0.37 and 0.88, respectively. Among several compounds affecting the PEP carboxylase activity, such as organic acids, amino acids, and sugar phosphates, at physiological pH (7.0 and 7.8), malate shows the strongest inhibition of a competitive character, its Ki being 2 mM. Also acidic amino acids strongly inhibit the enzyme activity, aspartate being more effective than glutamate. Glucose 6-phosphate and fructose 1,6-diphosphate markedly activate the enzyme. Both the inhibition by malate, aspartate and glutamate, and the activation by sugar phosphates rises considerably when pH is decreased from 7.8 to 7.0. Malonate scarcely affects the enzyme.     

Purification of lactoperoxidase from creek-water buffalo milk and investigation of kinetic and antibacterial properties

Water buffalo lactoperoxidase (WBLP) was purified with Amberlite CG 50 H+ resin, CM Sephadex C-50 ion-exchange chromatography, and Sephadex G-100 gel filtration chromatography from skim milk. All purification steps of the WBLP were shown with SDS-PAGE and Rz (A412/A280) controlled the purification degree of the enzyme. Rz value for the purified WBLP was 0.8. To determine purification steps and kinetic properties, the activity of enzyme was measured by using 2,2-azino-bis-(3-ethylbenzthiazoline-6 sulfonic acid) diammonium salt (ABTS) as a choromogenic substrate at pH=6. Km, Vmax, optimum pH, and optimum temperature for the WBLP were found by means of graphics for ABTS as substrates. Optimum pH and optimum temperature of the WBLP were 6 and 60 degrees C, respectively. Km value at optimum pH and optimum temperature for the WBLP was 0.82 mM. Vmax value at optimum pH and optimum temperature was 13.7 micromol/mL x min. Km value at optimum pH and 25 degrees C for the WBLP was 0.77 mM. Vmax value at optimum pH and 25 degrees C was 4.83 micromol/mL x min. The purified WBLP was found to have high antibacterial activity in a thiocynate-H2O2 medium for some pathogenic bacteria, such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginose, Shigella sonnei, Staphylococcus saphrophyticus, Staphylococcus epidermidis, and Shigella dysenteriae and compared with well known antibacterial substances such as tetracycline, penicillin, and netilmicine.     

Purification and characterization of thermostable beta-mannanase and alpha-galactosidase from Bacillus stearothermophilus.

Bacillus stearothermophilus secretes beta-mannanase and alpha-galactosidase enzymatic activities capable of hydrolyzing galactomannan substrates. Expression of the hemicellulase activities in the presence of locust bean gum was sequential, with mannanase activity preceding expression of alpha-galactosidase activity. The hemicellulase activities were purified to homogeneity by a combination of ammonium sulfate fractionation, gel filtration, hydrophobic interaction chromatography, and ion-exchange and chromatofocusing techniques. The purified beta-D-mannanase is a dimeric enzyme (162 kilodaltons) composed of subunits having identical molecular weight (73,000). Maximal activity did not vary between pH 5.5 and 7.5. The beta-D-mannanase activity exhibited thermostability, retaining nearly full activity after incubation for 24 h at 70 degrees C and pH 6.5. The enzyme displayed high specificity for galactomannan substrates, with no-secondary xylanase or cellulase activity detected. Hydrolysis of locust bean gum yielded short oligosaccharides compatible with an endo mode of substrate depolymerization. Initial rate velocities of the mannanase activity displayed substrate inhibition and yielded estimates for Vmax and Km of 455 +/- 60 U/mg and 1.5 +/- 0.3 mg/ml, respectively, at 70 degrees C and pH 6.5. The alpha-galactosidase activity corresponded to a trimeric enzyme (247 kilodaltons) having subunits of identical molecular weight (82,000). The alpha-galactosidase had maximal activity at pH 7 to 7.5 and retained full activity after 24 h of incubation at 60 degrees C. The enzyme had only limited activity on galactomannan substrates as compared with hydrolysis of p-nitrophenyl alpha-D-galactose. Kinetics of p-nitrophenyl alpha-D-galactose hydrolysis yielded linear reciprocal plots corresponding to Vmax and Km of 195 +/- 10 U/mg and 0.25 +/- 0.02 mM, respectively, at 60 degrees C and pH 7. The characterization of the mannanase activity is consistent with its potential use in enzymatic bleaching of softwood pulps.     

Purification and characterization of thermostable beta-mannanase and alpha-galactosidase from Bacillus stearothermophilus

Bacillus stearothermophilus secretes beta-mannanase and alpha-galactosidase enzymatic activities capable of hydrolyzing galactomannan substrates. Expression of the hemicellulase activities in the presence of locust bean gum was sequential, with mannanase activity preceding expression of alpha-galactosidase activity. The hemicellulase activities were purified to homogeneity by a combination of ammonium sulfate fractionation, gel filtration, hydrophobic interaction chromatography, and ion-exchange and chromatofocusing techniques. The purified beta-D-mannanase is a dimeric enzyme (162 kilodaltons) composed of subunits having identical molecular weight (73,000). Maximal activity did not vary between pH 5.5 and 7.5. The beta-D-mannanase activity exhibited thermostability, retaining nearly full activity after incubation for 24 h at 70 degrees C and pH 6.5. The enzyme displayed high specificity for galactomannan substrates, with no-secondary xylanase or cellulase activity detected. Hydrolysis of locust bean gum yielded short oligosaccharides compatible with an endo mode of substrate depolymerization. Initial rate velocities of the mannanase activity displayed substrate inhibition and yielded estimates for Vmax and Km of 455 +/- 60 U/mg and 1.5 +/- 0.3 mg/ml, respectively, at 70 degrees C and pH 6.5. The alpha-galactosidase activity corresponded to a trimeric enzyme (247 kilodaltons) having subunits of identical molecular weight (82,000). The alpha-galactosidase had maximal activity at pH 7 to 7.5 and retained full activity after 24 h of incubation at 60 degrees C. The enzyme had only limited activity on galactomannan substrates as compared with hydrolysis of p-nitrophenyl alpha-D-galactose. Kinetics of p-nitrophenyl alpha-D-galactose hydrolysis yielded linear reciprocal plots corresponding to Vmax and Km of 195 +/- 10 U/mg and 0.25 +/- 0.02 mM, respectively, at 60 degrees C and pH 7. The characterization of the mannanase activity is consistent with its potential use in enzymatic bleaching of softwood pulps.     

Purification of lactoperoxidase from bovine milk and investigation of the kinetic properties.

Lactoperoxidase (LPO) was purified from bovine milk using Amberlite CG 50 H+ resin, CM Sephadex C-50 ion-exchange chromatography, and Sephadex G-100 gel filtration chromatography. During the purification steps, the activity of enzyme was measured using 2,2'-azino-bis (3-ethylbenzthiazoline-6 sulfonic acid) diamonium salt (ABTS) as a chromogenic substrate at pH 6. Optimum pH and optimum temperature values for LPO were determined for ABTS, p-phenylendiamine, catechol, epinephrine, and pyrogallol as substrates, and then Km and Vmax values for the same substrate were obtained by means of Lineweaver-Burk graphics. The purification degree of the enzyme was controlled by SDS-PAGE and Rz (A412/A280) values. Km values, at optimum pH and 20 degrees C, were 0.197 mM, 0.063 mM, 0.64 mM, 25.2 mM, and 63.95 mM for p-phenylendiamine, ABTS, epinephrine, pyrogallol, and catechol, respectively. Vmax values, at optimum pH and 20 degrees C, were 3.5x10(-5) EU/mL, 4.0x10(-5) EU/mL, 5.8x10(-4) EU/mL, 8.4x10(-4) EU/mL, and 1.01x10(-3) EU/mL for the same substrates, respectively. p-Phenylendiamine was first found as a new substrate for LPO.     

Purification and general properties of pectin methyl esterase from Curvularia inaequalis NRRL 13884 in solid state culture using orange peels as an inducer

Pectin methyl esterase (PME) [E.C.3. 1.1.11] production by Curvularia inaequalis (Shear) Boedijn NRRL 13884 was investigated using solid-state culture. The highest level of extracellular pectin methyl esterase was detected with orange peels as an inducing substrate and as a sole carbon source. The enzyme was partially purified using Sephadex G-100 and DEAE-Cellulose column chromatography. It was purified about 40 fold with optimum activity at pH 4.4 and 45 degrees C. The enzyme was activated by Co++, Mg++, Na+, whereas it was slightly activated in the presence of Cu++, K+, Mn++, Zn++. On the other hand Ag++, Ca++ and Hg++ inhibited the activity of the enzyme. The Km was calculated to be 0.52 mM.     

Purification and properties of thiosulfate dehydrogenase from Acidithiobacillus thiooxidans JCM7814

A key enzyme of the thiosulfate oxidation pathway in Acidithiobacillus thiooxidans JCM7814 was investigated. As a result of assaying the enzymatic activities of thiosulfate dehydrogenase, rhodanese, and thiosulfate reductase at 5.5 of intracellular pH, the activity of thiosulfate dehydrogenase was measured as the key enzyme. The thiosulfate dehydrogenase of A. thiooxidans JCM7814 was purified using three chromatographies. The purified sample was electrophoretically homogeneous. The molecular mass of the enzyme was 27.9 kDa and it was a monomer. This enzyme had cytochrome c. The optimum pH and temperature of this enzyme were 3.5 and 35 degrees C. The enzyme was stable in the pH range from 5 to 7, and it was stable up to 45 degrees C. The isoelectric point of the enzyme was 8.9. This enzyme reacted with thiosulfate as a substrate. The Km was 0.81 mM.     

Wound-inducible pinene cyclase from grand fir: purification, characterization, and renaturation after SDS-PAGE.

The major wound-inducible monoterpene synthase (cyclase) of grand fir (Abies grandis) stems transforms geranyl pyrophosphate to both (-)-alpha-pinene (40%) and (-)-beta-pinene (60%). The enzyme was purified to apparent homogeneity by anion-exchange and hydrophobic interaction chromatography, coupled to discontinuous native polyacrylamide gel electrophoresis at neutral pH and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (also at neutral pH) followed by renaturation in 1% Tween 20 (polyoxyethylenesorbitan monolaurate). The renatured enzyme produced a mixture of isomeric pinenes from geranyl pyrophosphate identical to that generated by the native form. The protein exhibited a molecular weight of 63,000 by gel permeation chromatography and of 62,000 by denaturing gel electrophoresis, indicating that the monomer is active. The enzyme required Mn2+ (Km = 30 microM) for activity, exhibited a Km value of 6 microM for the substrate geranyl pyrophosphate, showed a pH optimum at 7.8 and temperature optimum at 42 degrees C, and was inhibited by pyrophosphate (I50 = 0.17 mM), orthophosphate (I50 = 51 mM), and alpha-pinene, as well as by the histidine-directed reagent diethylpyrocarbonate (I50 = 0.64 mM) and the cysteine-directed reagent p-hydroxymercuribenzoate (I50 = 1.9 microM). Although similar in many respects to constitutive monoterpene cyclases of herbaceous species, this inducible cyclase, the first enzyme of this type to be purified to homogeneity from a conifer, is distinguished by the relatively high pH optimum, and the strict specificity and high affinity for the divalent metal ion cofactor.     

Kinetics and effect of salts and polyamines on T4 polynucleotide ligase.

The kinetics of T4 polynucleotide ligase has been investigated at pH 8,20 degrees C and using the double-stranded DNA substrate (dA)n - [(dT)10]n/10. Double-reciprocal plots of initial rates vs substrate concentrations as well as product inhibition studies have indicated that the enzyme reacts according to a ping-pong mechanism. The overall mechanism was found to be non-processive. The true Km for the DNA substrate was 0.6 muM and that of ATP 100 muM. Several attempts were made to reverse the T4 polynucleotide ligase joining reaction using 32-p-labelled (dA)n - [(DT)40]n/40 as substrate. No breakdown of this DNA could be detected. The joining reaction was inhibited by high concentrations, i.e. above approximately 70mM, of salts such as KCl, NaCl, NH4Cl and CsCl. At a concentration of 200 mM almost 100% inhibition was observed. Polyamines also caused inhibition of the enzyme, the most efficient inhibitor being spermine followed by spermidine. At a concentration of 1 mM spermine, virtually no joining took place. Addition of salts or polyamines resulted in a large increase in the apparent Km for the DNA substrate whereas the apparent Km for ATP remained unchanged. It is suggested that the affinity of the enzyme for the DNA substrate is decreased in the presence of inhibiting agents.     

2-Hydroxyacid dehydrogenase from Haloferax mediterranei, a D-isomer-specific member of the 2-hydroxyacid dehydrogenase family

An NAD-dependent D-2-hydroxyacid dehydrogenase (EC 1.1.1.) was isolated and characterized from the halophilic Archaeon Haloferax mediterranei. The enzyme is a dimer with a molecular mass of 101.4 +/- 3.3 kDa. It is strictly NAD-dependent and exhibits its highest activity in 4 M NaCl. The enzyme is characterized by a broad substrate specificity 2-ketoisocaproate and 2-ketobutyrate being the substrates with the higher Vmax/Km. When pyruvate and 2-ketobutyrate were the substrates the optimal pH was acidic (pH 5) meanwhile for 2-ketoisocaproate maximum activity was achieved at basic pH between 7.5 and 8.5. The optimum temperature was 52 degrees C and at 65 degrees C there was a pronounced activity decrease. This new enzyme can be used for the production of D-2-hydroxycarboxylic acid.     

Biosynthesis of gallotannins: beta-glucogallin-dependent formation of 1,2,3,4,6-pentagalloylglucose by enzymatic galloylation of 1,2,3,6-tetragalloylglucose

An acyltransferase was detected in young leaves of pedunculate oak (Quercus robur) that catalyzed the formation of 1,2,3,4,6-penta-O-galloyl-beta-D-glucose, the common precursor of gallotannins and the related ellagitannins. This enzyme depended on beta-glucogallin (1-O-galloyl-beta-D-glucose) as acyl donor; 1,2,3,6-tetra-O-galloyl-beta-D-glucose was specifically required as acceptor molecule, whereas no reaction occurred with the 1,2,4,6-isomer of this substrate. The partially purified enzyme (Mr 260,000) was stable between pH 5.0 and 6.5; highest activities were observed at pH 6.3 and 40 degrees C. Km values of 2.3 and 1.0 mM, respectively, were determined for the substrates beta-glucogallin and tetragalloylglucose. In accordance with stoichiometric studies, the systematic name "beta-glucogallin: 1,2,3,6-tetra-O-galloylglucose 4-O-galloyltransferase" is proposed for this new enzyme.