Introducing transglycosylation activity in Bacillus licheniformis α-amylase by replacement of His235 with Glu

To understand the role of His and Glu in the catalytic activity of Bacillus licheniformis α-amylase (BLA), His235 was replaced with Glu. The mutant enzyme, H235E, was characterized in terms of its mode of action using labeled and unlabeled maltooctaose (Glc8). H235E predominantly produced maltotridecaose (Glc13) from Glc8, exhibiting high substrate transglycosylation activity, with K_m = 0.38 mM and k_cat/K_m = 20.58 mM⁻¹ s⁻¹ for hydrolysis, and K_m2 = 18.38 mM and k_cat2/K_m2 = 2.57 mM⁻¹ s⁻¹ for transglycosylation, while the wild-type BLA exhibited high hydrolysis activity exclusively. Glu235—located on a wide open groove near subsite +1—is likely involved in transglycosylation via formation of an α-1,4-glycosidic linkage and may recognize and stabilize the non-reducing end glucose of the acceptor molecule.     

Catechol 1,2-dioxygenase from the new aromatic compounds - Degrading Pseudomonas putida strain N6

This study aimed to characterization of catechol 1,2-dioxygenase from a Gram-negative bacterium, being able to utilize a wide spectrum of aromatic substrates as a sole carbon and energy source. Strain designated as N6, was isolated from the activated sludge samples of a sewage treatment plant at Bentwood Furniture Factory Jasienica, Poland. Morphology, physio-biochemical characteristics and phylogenetic analysis based on 16S rDNA sequence indicate that strain belongs to Pseudomonas putida. When cells of strain N6 grown on protocatechuate or 4-hydroxybenzoic acid mainly protocatechuate 3,4-dioxygenase was induced. The activity of catechol 1,2-dioxygenase was rather small. The cells grown on benzoic acid, catechol or phenol showed high activity of only catechol 1,2-dioxygenase. This enzyme was optimally active at 35 °C and pH 7.4. Kinetic studies showed that the value of K_m and V_max was 85.19 ??M and 14.54 ??M min⁻¹ respectively. Nucleotide sequence of gene encoding catechol 1,2-dioxygenase in strain N6 has 100% identity with catA genes from two P. putida strains. The deduced 301-residue sequence of enzyme corresponds to a protein of molecular mass 33.1 kDa. The deduced molecular structure of the catechol 1,2-dioxygenase from P. putida N6 was very similar and characteristic for the other intradiol dioxygenases.     

Defining a kinetic mechanism for l-DOPA 2,3 dioxygenase,a single-domain type I extradiol dioxygenase from Streptomyces lincolnensis

l-DOPA-2,3-dioxygenase from Streptomyces lincolnensis is a single domain type I extradiol dioxygenase of the vicinal oxygen chelate superfamily and catalyzes the second step in the metabolism of the propylhygric acid moiety of the antibiotic, lincomycin. In this report, the kinetic mechanism of l-DOPA dioxygenase is interrogated using stopped-flow in order to determine microscopic rate constants. Pre-steady state, progress curve and steady-state data were combined in a global kinetic analysis using KinTek Explorer in order to define and constrain a kinetic model for the type I l-DOPA dioxygenase. The data are best described by a four step mechanism, in which the cyclization of the enzymatic product is not enzyme catalyzed.     

Interaction of Fe(III) with herbicide-carboxylato ligands. Di-, tri- and tetra-nuclear compounds: Structure, antimicrobial study and DNA interaction

The iron complexes with the phenoxyalkanoic acids 2,3-D = 2,3-dichlorophenoxyacetic acid, 3,4-D = 3,4-dichlorophenoxyacetic acid, 2,4,5-T = 2,4,5-trichlorophenoxyacetic acid, and mcpa = 2-chloro-4-methyl-phenoxyacetic acid, in the presence or not of a nitrogen donor heterocyclic ligand, py = pyridine, bipy = 2,2′ bipyridine, phen = 1,10-phenanthroline, were prepared and characterized.The interaction of Fe(III) with phenoxyalkanoic acids and bipy or phen leads to dinuclear neutral complexes, while the presence of py favors tetranuclear neutral forms. The crystal structures of [Fe₂OCl₂(mcpa)₂(bipy)₂] · 0.25(bipy) · 0.8MeCN (1a), and {[Fe₄O₂(mcpa)₆Cl₂(py)₄] · 2MeCN} (3a), have been determined. DNA-Fe(III) complex interaction studies suggest that iron complexes promote the hydrolytic cleavage of double stranded DNA that seems to be oxygen independent, while pDNA shows cross-linking with many molecules of the iron clusters. Antibacterial screening data showed that the presence of chelating agents, bipy or phen, increased the efficiency of iron complexes.     

Kynurenine Pathway Enzymes in Brain: Responses to Ischemic Brain Injury Versus Systemic Immune Activation

Accumulation of l -kynurenine and quinolinic acid (QUIN) in the brain occurs after either ischemic brain injury or after systemic administration of pokeweed mitogen. Although conversion of l -[¹³C₆]tryptophan to [¹³C₆]-QUIN has not been demonstrated in brain either from normal gerbils or from gerbils given pokeweed mitogen, direct conversion in brain tissue does occur 4 days after transient cerebral ischemia. Increased activities of enzymes distal to indoleamine-2,3-dioxygenase may determine whether l -kynurenine is converted to QUIN. One day after 10 min of cerebral ischemia, the activities of kynureninase and 3-hydroxy-3,4-dioxygenase were increased in the hippocampus, but local QUIN levels and the activities of the indoleamine-2,3-dioxygenase and kynurenine-3-hydroxylase were unchanged. By days 2 and 4 after ischemia, however, the activities of all of these enzymes in the hippocampus as well as QUIN levels were significantly increased. Kynurenine aminotransferase activity in the hippocampus was unchanged on days 1 and 2 after ischemia but was decreased on day 4, at a time when local kynurenic acid levels were unchanged. A putative precursor of QUIN, [¹³C₆]anthranilic acid, was not converted to [¹³C₆]-QUIN in the hippocampus of either normal or 4-day postischemic gerbils. Gerbil macrophages stimulated by endo-toxin in vitro converted l -[¹³C₆]tryptophan to [¹³C_e]QUIN. Kinetic analysis of kynurenine-3-hydroxylase activity in the cerebral cortex of postischemic gerbils showed that V_max increased, without changes in K_m. Systemic administration of pokeweed mitogen increased indoleamine-2,3-dioxygenase and kynureninase activities in the brain without significant changes in kynurenine-3-hydroxylase or 3-hydroxyanthranilate-3,4-dioxygenase activities. Increases in kynurenine-3-hydroxylase activity, in conjunction with induction of indoleamine-2,3-dioxygenase, kynureninase, and 3-hydroxyanthranilate-3,4-dioxygenase in macro-phage infiltrates at the site of brain injury, may explain the ability of postischemic hippocampus to convert l -[¹³C₆]tryptophan to [¹³C₆]QUIN.     

Novel enzymatic activity derived from the Semliki Forest virus capsid protein

The N-terminal segment of the Semliki Forest virus polyprotein is an intramolecular serine protease that cleaves itself off after the invariant Trp267 from a viral polyprotein and generates the mature capsid protein. After this autoproteolytic cleavage, the free carboxylic group of Trp267 interacts with the catalytic triad (His145, Asp167 and Ser219) and inactivates the enzyme. We have deleted the last 1-7 C-terminal residues of the mature capsid protease to investigate whether removal of Trp267 regenerates enzymatic activity. Although the C-terminally truncated polypeptides do not adopt a defined three-dimensional structure and show biophysical properties observed in natively unfolded proteins, they efficiently catalyse the hydrolysis of aromatic amino acid esters, with higher catalytic efficiency for tryptophan compared to tyrosine esters and k_cat/K_M values up to 5 × 10⁵ s⁻¹ M⁻¹. The enzymatic mechanism of these deletion variants is typical of serine proteases. The pH enzyme activity profile shows a pK_a1 = 6.9, and the Ser219Ala substitution destroys the enzymatic activity. In addition, the fast release of the first product of the enzymatic reaction is followed by a steady-state second phase, indicative of formation and breakdown of a covalent acyl-enzyme intermediate. The rates of acylation and deacylation are k₂ = 4.4±0.6 s⁻¹ and k₃ = 1.6±0.5 s⁻¹, respectively, for a tyrosine derivative ester substrate, and the amplitude of the burst phase indicates that 95% of the enzyme molecules are active. In summary, our data provide further evidence for the potential catalytic activity of natively unfolded proteins, and provide the basis for engineering of alphavirus capsid proteins towards hydrolytic enzymes with novel specificities.     

2,3-Dihydroxy-quinoxaline induces ATPase activity of Herpes Simplex Virus thymidine kinase

2,3-Dihydroxy-quinoxaline, a small molecule that promotes ATPase catalytic activity of Herpes Simplex Virus thymidine kinase (HSV-TK), was identified by virtual screening. This compound competitively inhibited HSV-TK catalyzed phosphorylation of acyclovir with K_i = 250 μM (95% CI: 106–405 μM) and dose-dependently increased the rate of the ATP hydrolysis with K_M = 112 μM (95% CI: 28–195 μM). The kinetic scheme consistent with this experimental data is proposed.     

The contribution of SNAT1 to system A amino acid transporter activity in human placental trophoblast

System A-mediated amino acid transport across the placenta is important for the supply of neutral amino acids needed for fetal growth. All three system A subtypes (SNAT1, 2, and 4) are expressed in human placental trophoblast suggesting there is an important biological role for each. Placental system A activity increases as pregnancy progresses, coinciding with increased fetal nutrient demands. We have previously shown SNAT4-mediated system A activity is higher in first trimester than at term, suggesting that SNAT1 and/or SNAT2 are responsible for the increased system A activity later in gestation. However, the relative contribution of each subtype to transporter activity in trophoblast at term has yet to be evaluated. The purpose of this study was to identify the predominant subtype of system A in cytotrophoblast cells isolated from term placenta, maintained in culture for 66 h, by: (1) measuring mRNA expression of the three subtypes and determining the Michaelis-Menten constants for uptake of the system A-specific substrate, ¹⁴C-MeAIB, (2) investigating the contribution of SNAT1 to total system A activity using siRNA. Results: mRNA expression was highest for the SNAT1 subtype of system A. Kinetic analysis of ¹⁴C-MeAIB uptake revealed two distinct transport systems; system 1: K_m = 0.38 ± 0.12 mM, V_max = 27.8 ± 9.0 pmol/mg protein/20 min, which resembles that reported for SNAT1 and SNAT2 in other cell types, and system 2: K_m = 45.4 ± 25.0 mM, V_max = 1190 ± 291 pmol/mg protein/20 min, which potentially represents SNAT4. Successful knockdown of SNAT1 mRNA using target-specific siRNA significantly reduced system A activity (median 75% knockdown, n = 7). Conclusion: These data enhance our limited understanding of the relative importance of the system A subtypes for amino acid transport in human placental trophoblast by demonstrating that SNAT1 is a key contributor to system A activity at term.     

The Metalloregulatory Zinc Site in Streptococcus pneumoniae AdcR, a Zinc-activated MarR Family Repressor

Streptococcus pneumoniae D39 AdcR (adhesin competence repressor) is the first metal-sensing member of the MarR (multiple antibiotic resistance repressor) family to be characterized. Expression profiling with a ΔadcR strain grown in liquid culture (brain-heart infusion) under microaerobic conditions revealed upregulation of 13 genes, including adcR and adcCBA, encoding a high-affinity ABC uptake system for zinc, and genes encoding cell-surface zinc-binding pneumococcal histidine triad (Pht) proteins and AdcAII (Lmb, laminin binding). The ΔadcR, H108Q and H112Q adcR mutant allelic strains grown in 0.2 mM Zn(II) exhibit a slow-growth phenotype and an approximately twofold increase in cell-associated Zn(II). Apo- and Zn(II)-bound AdcR are homodimers in solution and binding to a 28-mer DNA containing an adc operator is strongly stimulated by Zn(II) with K_DNA-Zn = 2.4 × 10⁸ M^- 1 (pH 6.0, 0.2 M NaCl, 25 °C). AdcR binds two Zn(II) per dimer, with stepwise Zn(II) affinities K_Zn1 and K_Zn2 of ≥ 10⁹ M^- 1 at pH 6.0 and ≥ 10¹² M^- 1 at pH 8.0, and one to three lower affinity Zn(II) depending on the pH. X-ray absorption spectroscopy of the high-affinity site reveals a pentacoordinate N/O complex and no cysteine coordination, the latter finding corroborated by wild type-like functional properties of C30A AdcR. Alanine substitution of conserved residues His42 in the DNA-binding domain, and His108 and His112 in the C-terminal regulatory domain, abolish high-affinity Zn(II) binding and greatly reduce Zn(II)-activated binding to DNA. NMR studies reveal that these mutants adopt the same folded conformation as dimeric wild type apo-AdcR, but fail to conformationally switch upon Zn(II) binding. These studies implicate His42, His108 and H112 as metalloregulatory zinc ligands in S. pneumoniae AdcR.     

A fluorescence assay for tetradecyltrimethylammonium mono-oxygenase activity that catalyzes the cleavage of the C-N bond with the production of trimethylamine

This article describes a simple fluorescence method for the determination of tetradecyltrimethylammonium mono-oxygenase (TTAB mono-oxygenase) activity involving N-dealkylation of tetradecyltrimethylammonium bromide with concomitant production of trimethylamine (TMA). Activity was determined by measuring the formation of TMA using the morin reagent and aluminum (Al). Morin reacts with Al to form a fluorescent complex, Al-morin. In the presence of TMA, Al is tightly associated with TMA and cannot be sequestered by morin, thus providing evidence for formation of the Al-TMA complex. The concentration of TMA is estimated by calibration graphs constructed by plotting the fluorescence intensity of the Al-morin complex versus TMA concentration. The fluorescence intensities of the Al-morin complexes quenched by TMA are linearly dependent on both the time of the TTAB mono-oxygenase reaction and the amount of protein used in the reaction. The kinetic behavior is characterized by K_0.5 = 4.26 × 10⁻⁴ M, and the apparent Hill coefficient (n_app) = 2.24. These values are both comparable to those determined by GC-MS (K_0.5 = 4.41 × 10⁻⁴ M and n_app = 2.35). The advantages of this assay include rapid and efficient implementation and potential employment for routine accurate determinations of TTAB mono-oxygenase activity over a wide range of substrate concentrations.     

Development of a scintillation proximity assay binding method for the human 5-hydroxytryptamine 6 receptor using intact cells

We describe the first validated scintillation proximity assay (SPA) binding method for quantitation of ³H-labeled d-lysergic acid diethylamide (LSD) binding to recombinant human 5-hydroxytryptamine 6 (5-HT₆) receptors expressed in Chinese hamster ovary (CHO)-Dukx and HeLa cells. The assay was developed using intact cells as a receptor source because membrane fractions derived from these cells failed to discern specific binding from a high level of nonspecific binding. The pharmacological binding profile of seven 5-HT₆ agonists and antagonists using intact CHO-Dukx/5-HT₆ cells in the SPA format was similar to data obtained from a filtration binding assay using HeLa/5-HT₆ membranes. K_i values and rank order of potencies obtained in the SPA format were consistent with published filtration data as follows: SB-271046 (K_i = 1.9 nM) > methiothepin (K_i = 6.2 nM) > mianserin (K_i = 74.3 nM) > 5-methoxytryptamine (5-MeOT, K_i = 111 nM) > 5-HT (K_i = 150 nM) > ritanserin (K_i = 207 nM) > 5-carboxamidotryptamine (5-CT, K_i = 704 nM). Additional evaluation with four antipsychotics demonstrated strong agreement with previous literature reports. A high specific binding signal and low assay variability, as determined by Z′ = 0.81 ± 0.017, make the SPA format amenable to automation and higher throughput; hence, this assay can be a viable alternative to the more labor-intensive filtration and centrifugation methods.     

Nitrilotris(methylenephosphonates) in aqueous solution and solid state - dilatometric, potentiometric and NMR investigations

Dissociation and alkali complex formation equilibria of nitrilotris(methylenephosphonic acid) (NTMP, H₆L) have been studied by dilatometric, potentiometric and ³¹P NMR-controlled titrations. Dilatometry indicated the formation of alkali complexes ML (M=Li, Na, K, Rb, Cs) at high pH with a stability decreasing from Li to Cs. An efficient combination of potentiometric and NMR methods confirmed two types of alkali metal complexes MHL and ML. Stability constants for the equilibria following M⁺ + HL⁵⁻ ? MHL⁴⁻ and M⁺ + L⁶⁻ ? ML⁵⁻, respectively, were determined: logK_NaHL=1.08(0.07), logK_KHL=0.86(0.08), logK_NaL=2.24(0.03). Systematic errors are introduced by using alkali metal hydroxides as titrants for routine potentiometric determinations of dissociation constants pK_a5^app and pK_a6^app. Correction formulae were derived to convert actual dissociation constants pK_a into apparent dissociation constants pK_a^app (or vice versa). The actual dissociation constants were found: pK_a5(H₂L⁴⁻ ? H⁺ + HL⁵⁻)=7.47(0.03) and pK_a6(HL⁵⁻ ? H⁺ + L⁶⁻)=14.1(0.1). The anisotropy of ³¹P chemical shifts of salts M_nH_6 − nL (M=Li, Na, n=0-5) is more sensitive towards titration (n) than isotropic solution state chemical shifts.     

Oxidation of thiocyanate by iron(V) in alkaline medium

The oxidation of thiocyanate by iron(V) (Fe(V)) was studied as a function of pH in alkaline solutions by a premix pulse radiolysis technique. The rates decrease with an increase in pH. The rate law for the oxidation of SCN⁻ by Fe(V) was obtained as −d[Fe(V)]/dt = k₁₀{[H⁺]²/([H⁺]² + K₂[H⁺] + K₂K₃)}[Fe(V)][SCN⁻], where k₁₀ = 5.72 ± 0.19 × 10⁶ M⁻¹ s⁻¹, pK₂ = 7.2, and pK₃ = 10.1. The reaction precedes via a two-electron oxidation, which converts Fe(V) to Fe(III). Thiocyanate reacts approximately 10³× faster with iron(V) than does with iron(VI).     

High level expression of a novel β-mannanase from Chaetomium sp. exhibiting efficient mannan hydrolysis

A novel β-mannanase gene (CsMan5A) was cloned from Chaetomium sp. CQ31 and expressed in Pichia pastoris. It had an open reading frame of 1251 bp encoding 416 amino acids and contained two introns. The deduced amino acid sequence shared the highest similarity (73%) with the β-mannanase from Emericella nidulans and belongs to glycosyl hydrolase family 5. The recombinant β-mannanase (CsMan5A) was secreted at extremely high levels of 50,030 U mL⁻¹ and 6.1 mg mL⁻¹ in high cell density fermentor. The purified enzyme was optimally active at pH 5.0 and 65 °C and displayed broad pH stability (pH 5.0-11.0) and exhibited specificity towards locust bean gum (K_m = 3.1 mg mL⁻¹), guar gum (K_m = 9.3 mg mL⁻¹) and konjac powder (K_m = 10.5 mg mL⁻¹). It efficiently degraded mannan polysaccharides into mannose and mannooligosacccharides, and also hydrolyzed mannotriose and mannotetraose. These properties make CsMan5A highly useful in food, feed and paper/pulp industries.     

Modeling the Zn and Cd metalation mechanism in mammalian metallothionein 1a

Mammalian metallothioneins (MTs) are a family of small cysteine rich proteins believed to have a number of physiological functions, including both metal ion homeostasis and toxic metal detoxification. Mammalian MTs bind 7 Zn²⁺ or Cd²⁺ ions into two distinct domains: an N-terminal β-domain that binds 3 Zn²⁺ or Cd²⁺, and a C-terminal α-domain that binds 4 Zn²⁺ or Cd²⁺. Although stepwise metalation to the saturated M₇-MT (where M = Zn²⁺ or Cd²⁺) species would be expected to take place via a noncooperative mechanism involving the 20 cysteine thiolate ligands, literature reports suggest a cooperative mechanism involving cluster formation prior to saturation of the protein. Electrospray ionization mass spectrometry (ESI-MS) provides this sensitivity through delineation of all species (M_n-MT, n = 0-7) coexisting at each step in the metalation process. We report modeled ESI-mass spectral data for the stepwise metalation of human recombinant MT 1a (rhMT) and its two isolated fractions for three mechanistic conditions: cooperative (where the binding affinities are: K₁ < K₂ < K₃ < ··· < K₇), weakly cooperative (where K₁ = K₂ = K₃ = ··· = K₇), and noncooperative, (where K₁ > K₂ > K₃ > ··· > K₇). Detailed ESI-MS metalation data of human recombinant MT 1a by Zn²⁺ and Cd²⁺ are also reported. Comparison of the experimental data with the predicted mass spectral data provides conclusive evidence that metalation occurs in a noncooperative fashion for Zn²⁺ and Cd²⁺ binding to rhMT 1a.     

Characterization of Ca and phosphocholine interactions with C-reactive protein using a surface plasmon resonance biosensor

The interactions between Ca²⁺ and C-reactive protein (CRP) have been characterized using a surface plasmon resonance (SPR) biosensor. The protein was immobilized on a sensor chip, and increasing concentrations of Ca²⁺ or phosphocholine were injected. Binding of Ca²⁺ induced a 10-fold higher signal than expected from the molecular weight of Ca²⁺. It was interpreted to result from the conformational change that occurs on binding of Ca²⁺. Two sites with different characteristics were distinguished: a high-affinity site with K_D = 0.03 mM and a low-affinity site with K_D = 5.45 mM. The pH dependencies of the two Ca²⁺ interactions were different and enabled the assignment of the different sites in the three-dimensional structure of CRP. There was no evidence for cooperativity in the phosphocholine interaction, which had K_D = 5 μM at 10 mM Ca²⁺. SPR biosensors can clearly detect and quantify the binding of very small molecules or ions to immobilized proteins despite the theoretically very low signals expected on binding, provided that significant conformational changes are involved. Both the interactions and the conformational changes can be characterized. The data have important implications for the understanding of the function of CRP and suggest that Ca²⁺ is an efficient regulator under physiological conditions.     

A novel NADP-dependent dehydrogenase activity for 7α/β- and 11β-hydroxysteroids in human liver nuclei: A third 11β-hydroxysteroid dehydrogenase

Human tissue from uninvolved liver of cancer patients was fractionated using differential centrifugation and characterized for 11βHSD enzyme activity against corticosterone, dehydrocorticosterone, 7α- and 7β-hydroxy-dehydroepiandrosterone, and 7-oxo-dehydroepiandrosterone. An enzyme activity was observed in nuclear protein fractions that utilized either NADP⁺ or NAD⁺, but not NADPH and NADH, as pyridine nucleotide cofactor with K_m values of 12 ± 2 and 390 ± 2 μM, compared to the K_m for microsomal 11βHSD1 of 43 ± 8 and 264 ± 24 μM, respectively. The K_m for corticosterone in the NADP⁺-dependent nuclear oxidation reaction was 102 ± 16 nM, compared to 4.3 ± 0.8 μM for 11βHSD1. The K_cat values for nuclear activity with NADP⁺ was 1687 nmol/min/mg/μmol, compared to 755 nmol/min/mg/μmol for microsomal 11βHSD1 activity. Inhibitors of 11βHSD1 decreased both nuclear and microsomal enzyme activities, suggesting that the nuclear activity may be due to an enzyme similar to 11βHSD Type 1 and 2.     

Synthesis and characterization of [Fe(III)(qsal)2][M(III)(pds)2] (M = Cu, Au)

Salts of the Fe(III) spin crossover cation [Fe^III(qsal)₂]⁺ (qsalH = N-(8-quinolyl)salicylaldimine) and monoanions [M^III(pds)₂]⁻ (M = Cu, Au; pds = pirazine-2,3-diselenolate) with formula [Fe^III(qsal)₂][M^III(pds)₂] were prepared and characterized by single crystal X-ray diffraction and magnetic measurements. These two salts present magnetic properties essentially due to the Fe^III centres in the high-spin state (S = 5/2), and do not have any spin transition.     

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.