Characteristics of a New Catechol 1,2-Oxygenase from Trichosporon cutaneum WY 2-2

Successive feeding of phenol at concentrations of less than 5.5 mM into a thick suspension of Trichosporon cutaneum WY 2-2 precultured in MPY-medium resulted in a high yield (approximately 28.7 g wet cells/liter) of intact cells capable of decomposing phenol actively (3.7 μmol/min/g of wet cells).

The effects of pH and additions of ethanol and 2-mercaptoethanol were tested on the stability of crude extracts from the strain grown on phenol. The crude extracts were stable at a pH range of 7.6 and 8.3, and were stable for 35 days when 10% ethanol and 5 mM 2-mercaptoethanol were added.

A highly purified preparation of catechol 1,2-oxygenase was obtained from strain WY 2-2 grown on phenol. The purified enzyme was homogeneous on polyacrylamide disc-gel electrophoresis. The enzyme had a molecular weight of about 105,000 and gave rise to subunits of molecular weight of 35,000 by SDS gel electrophoresis. Therefore, the enzyme appears to be a trimer of subunits with identical molecular weight. The Michaelis constants were 9.0 μM for catechol and 6.8 μM for 4-methylcatechol. The enzyme exhibited higher activities towards 4-methylcatechol and hydroxyquinol than towards catechol, and had essentially the same substrate specificity as the crude extracts. 4-Methylcatechol completely inhibited the enzyme activity towards catechol.     

Hydrolysis of fucosyl-GM1 ganglioside by purified pellet-associated human brain and human liver alpha-L-fucosidases without activator proteins or detergents.

Pellet-associated human brain alpha-L-fucosidase was solubilized with 0.5% (w/v) Triton X-100 and purified by affinity chromatography on agarose-6-aminohexanoyl-fucosamine resin. The procedure resulted in a 290,000-fold purification, a 58% yield and a final specific activity of 11,500 nmol/min per mg of protein. Isoelectric focusing indicated that all six major isoforms (with pI values between 4.1 and 5.3) present in crude brain pellet preparations were purified by the affinity technique. SDS/PAGE indicated the presence of one subunit (54 kDa) and a minor protein band at 67 kDa, which presumably is a contaminant since it was not immunoreactive on Western blotting. The pH optimum of the brain enzyme and its apparent Km for the synthetic substrate 4-methylumbelliferyl alpha-L-fucopyranoside were 5.5 and 0.07 mM respectively. Pellet-associated human brain and liver alpha-L-fucosidases were both capable of hydrolysing fucosyl-GM1 ganglioside without activator proteins or detergents. Linear hydrolysis rates were found only for short incubation times (1-5 min). Optimal enzymic activity at 37 degrees C was found at pH 3.4 for both alpha-L-fucosidases, with no activity at pH values above 4.0.     

Hemocyanin-derived phenoloxidase activity in the spiny lobster Panulirus argus (Latreille, 1804)

Hemocyanin and phenoloxidase belong to the type-3 copper protein family, sharing a similar active center whereas performing different roles. In this study, we demonstrated that purified hemocyanin (450 kDa) from the spiny lobster Panulirus argus shows phenoloxidase activity in vitro after treatment with trypsin, chymotrypsin and SDS (0.1% optimal concentration), but it is not activated by sodium perchlorate or isopropanol. The optimal pHs of the SDS-activated hemocyanin were 5.5 and 7.0. Hemocyanin from spiny lobster behaves as a catecholoxidase. Kinetic characterization using dopamine, L-DOPA and catechol shows that dopamine is the most specific substrate. Catechol and dopamine produced substrate inhibition above 16 and 2 mM respectively. Mechanism-based inhibition was also evidenced for the three substrates, being less significant for L-DOPA. SDS-activated phenoloxidase activity is produced by the hexameric hemocyanin. Zymographic analysis demonstrated that incubation of native hemocyanin with trypsin and chymotrypsin, produced bands of 170 and 190 kDa respectively, with intense phenoloxidase activity. Three polypeptide chains of 77, 80 and 89 kDa of hemocyanin monomers were identified by SDS-PAGE. Monomers did not show phenoloxidase activity induced by SDS or partial proteolysis.     

Characterization of polyphenol oxidase in coffee

Polyphenol oxidase (PPO) was characterized in partially purified extracts of leaves (PPO-L) and fruit endosperm (PPO-E) of coffee (Coffea arabica L.). PPO activity was higher in early developmental stages of both leaves and endosperm of fruits. Wounding or exposure of coffee leaves to methyl jasmonate increased PPO activity 1.5-4-fold. PPO was not latent and was not activated by protease treatment. PPO activity was stimulated 10-15% with sodium dodecyl sulphate (SDS) at 0.35-1.75 mM, but at higher concentrations activities were similar to the control samples, without detergent. Prolonged incubation of extracts with trypsin or proteinase K inhibited PPO activity but pepsin had no effect. Inhibition of PPO with proteinase K was increased in the presence of SDS. PPO activity from both tissues was optimal at pH 6-7 and at an assay temperature of 30 degrees C. Activity was highest with chlorogenic acid as substrate with a Km of 0.882 mM (PPO-L) and 2.27 mM (PPO-E). Hexadecyl trimethyl-ammonium bromide, polyvinylpyrrolidone 40. cinnamic acid and salicylhydroxamic acid inhibited PPO from both tissues. Both enzymes were inactivated by heat but the activity in endosperm extracts was more heat labile than that from leaves. The apparent Mr determined by gel filtration was 46 (PPO-L) and 50 kDa (PPO-E). Activity-stained SDS polyacrylamide gel electrophoresis (PAGE) gels and western blots probed with PPO antibodies suggested the existence of a 67 kDa PPO which is susceptible to proteolytic cleavage that generates a 45 kDa active form.     

Purification and characterization of ferulate and p-coumarate decarboxylase from Bacillus pumilus.

Bacillus pumilus PS213 isolated from bovine ruminal fluid was able to transform ferulic acid and p-coumaric acid to 4-vinylguaiacol and 4-vinylphenol, respectively, by nonoxidative decarboxylation. The enzyme responsible for this activity has been purified and characterized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude extract from a culture induced by ferulic acid or p-coumaric acid shows three bands that are not present in the crude extract of an uninduced culture, while the purified enzyme shows a single band of 23 kDa; the molecular mass calculated by size exclusion chromatography is 45 kDa. Enzyme activity is optimal at 37 degrees C and pH 5.5 and is not enhanced by any cation. Kinetic studies indicated a Km of 1.03 mM and a Vmax of 0.19 mmol.min-1/mg.liter-1 for ferulic acid and a Km of 1.38 mM and a Vmax of 0.22 mmol.min-1/mg.liter-1 for p-coumaric acid.     

Purification and characterization of a lactonase from Burkholderia sp. R-711, that hydrolyzes (R)-5-oxo-2-tetrahydrofurancarboxylic acid

A lactonase hydrolyzing (R)-5-oxo-2-tetrahydrofurancarboxylic acid to D-alpha-hydroxyglutaric acid was purified 170-fold with 2% recovery to near homogeneity from crude extracts of Burkholderia sp. R-711, which had been isolated as a bacterium able to assimilate (R)-5-oxo-2-tetrahydrofurancarboxylic acid. The molecular mass was estimated to be 33 kDa by gel filtration. The purified preparation migrated as a single band of molecular mass 38 kDa upon SDS-PAGE. The maximum activity was observed at pH 7.0-8.0 and 35-40 degrees C. The enzyme required no added cofactors or metal ions; the activity was inhibited to 60-100% by SH-blocking reagents, but was not affected by metal-chelating reagents. The enzyme showed lower activity and affinity toward (S)-5-oxo-2-tetrahydrofurancarboxylic acid, but did not act on other natural and synthetic lactones tested.     

Isolation and characterization of novel tyrosinase from Laceyella sacchari

We here describe the isolation and characterization of a tyrosinase from a newly isolated soil bacterium. 16S rDNA sequence analysis revealed that the bacterium most probably belongs to the species Laceyella sacchari (Ls) ( > 99.9 % identity). The tyrosinase extracellular enzymatic activity was induced in the presence of L-methionine and CuSO4. The crude enzyme was first purified by centrifugation followed by ammonium sulphate precipitation and ultrafiltration. After removal of a brown pigment, probably melanin, a purified enzyme was obtained by further separation of the crude protein mixture using size exclusion chromatography. Some 10.5 mg of pure tyrosinase (LsTyr) was isolated with a molecular mass of 30 910 Da, based on MALDI mass spectrometry. Together with the observed enzymatic activity, N-terminal chemical sequence analysis confirmed that the isolated enzyme is homologous to other tyrosinases. The kinetic parameters for the diphenol substrates L-DOPA and dopamine and for the monophenol substrate L-tyrosine were determined to be KM = 4.5 mM , 1.5 mM and 0.055 mM, and kcat/KM = 261.5 mM-1 s -1 , 30.6 mM-1 s-1 and 56.3 mM-1 s-1, respectively. Maximal activities of the purified enzyme were found to occur at pH 6.8.     

Activity, isoenzymes and purity of mushroom tyrosinase in commercial preparations

Several lots of commercial tyrosinase preparations were examined with regard to their enzyme activity, isoenzyme composition and purity. Enzyme activity toward catechol, -dopa and tyrosine showed significant variations from lot to lot and activation by SDS. Distribution of isoenzyme forms also varied from lot to lot. Comparisons of electrophoretic and isoelectric focusing protein profiles showed considerable differences and distributions of the proteins in each sample. Tyrosinase appeared to be a minor component in each preparation when compared to a partially purified enzyme. Investigators using commercial tyrosinase should exercise caution in interpreting data due to the presence of different isoenzyme forms, their distribution in various lots, and the presence of numerous other proteins.     

Purification and specificity of two alpha-glucosidase isoforms of the parasitic protist Trichomonas vaginalis

Two isoforms of alpha-glucosidase were purified from the parasitic protist Trichomonas vaginalis. Both consisted of 103 kDa subunits, but differed in pH optimum and substrate specificity. Isoform 1 had a pH optimum around 4.5 and negligible activity on glucose oligomers other than maltose, while isoform 2 with a pH optimum of 5.5 hydrolyzed also such substrates at considerable rates. Neither had activity on glycogen or starch. Isoform 1 had a specific activity for hydrolysis of maltose of 30 U/mg protein and isoform 2 101 U/mg protein. The Km values were 0.4 mM and 2.0 mM, respectively. Isoform 2 probably corresponds to the activity detected on the cell surface.     

Effect of CaCl2 as activity stabilizer on purification of heparinase I from Flavobacterium heparinum

Heparinase I has been purified from F. heparinum by a novel scheme with 10mM CaCl(2) added in crude extracts of cells. The enzyme was purified to apparent homogeneity through ammonium sulfate precipitation, Octyl-Sepharose chromatography, CM-52 chromatography, SP-650 chromatography, and Sephadex G-100 gel filtration chromatography. The specific activity of the purified enzyme was 90.33 U/mg protein with a purification fold of 185.1. The yield was 17.8%, which is higher than any previous scheme achieved. The molecular weight of the purified enzyme was 43 kDa with a pI of 8.5. It has an activity maximum at pH range of 6.4-7.0 and 41 degrees C. CaCl(2) is a good stabilizer of the purified enzyme in liquid form toward either storaging at 4 degrees C or freezing-thawing.     

A wounding-induced PPO from cowpea (Vigna unguiculata) seedlings

Polyphenol oxidases (PPO) are induced in cowpea plants by wounding. The highest activity levels were detected 48h after this stimulus in both wounded and neighbor-to-wounded unifoliates of cowpea seedlings; the increase of activity was in the order of 13 to 15-fold, respectively, in comparison to control unifoliates. Multiple molecular forms of active PPO (Mrs 58, 73 and congruent with220kDa) were detected by partially denaturing SDS-PAGE. Wounding-induced cowpea PPO were extracted and purified through (NH(4))(2)SO(4) precipitation and ion-exchange chromatography. The effects of substrate specificity, pH, thermal stability and sensitivity to various inhibitors - resorcinol, EDTA, sodium azide and tropolone - of partially purified soluble PPO were investigated. Purified wounding-induced cowpea PPO (wicPPO) showed the highest activities towards 4-methylcatechol (K(m)=9.86mM, V(max)=24.66 EU [DeltaAmin(-1)]) and catechol (K(m)=3.44mM, V(max)=6.64 EU [DeltaAmin(-1)]); no activity was observed towards l-tyrosine, under the assay conditions used. The optimum pH for wound-induced cowpea PPO was 6.0 with 4-methylcatechol as substrate. The enzyme was optimally activated by 10 mM SDS and was highly stable even after 5 min at 80 degrees C. The most effective inhibitor was tropolone, whereas addition of 10mM of resorcinol, EDTA and sodium azide were able to reduce PPO activities by 40%, 15% and 100%, respectively.     

Isolation of a new heterodimeric lectin with mitogenic activity from fruiting bodies of the mushroom Agrocybe cylindracea

From the dried fruiting bodies of the mushroom Agrocybe cylindracea a heterodimeric lectin with a molecular weight of 31.5 kDa and displaying high hemagglutinating activity was isolated. The molecular weights of its subunits were 16.1 kDa and 15.3 kDa respectively. The larger and the smaller subunits resembled Agaricus bisporus lectin and fungal immunomodulatory protein from Volvariella volvacea respectively in N-terminal sequence. The lectin was adsorbed on DEAE-cellulose in 10 mM Tris-HCl buffer (pH 7.4) and was eluted by the same buffer containing 150 mM NaCl. It was adsorbed on SP-Sepharose in 10 mM NH4OAc (pH 4.5) and eluted by approximately 0.19 M NaCl in the same buffer. The lectin was obtained in a purified form after the mushroom extract had been subjected to (NH4)2SO4 precipitation and the two aforementioned ion exchange chromatographic steps. The lectin exhibited potent mitogenic activity toward mouse splenocytes. The hemagglutinating activity of the lectin was inhibited by lactose, sialic acid and inulin.     

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.     

Purification of Multiple Forms of Glutathione Reductase from Pea (Pisum sativum L.) Seedlings and Enzyme Levels in Ozone-Fumigated Pea Leaves

Glutathione reductase was purified from pea seedlings using a procedure that included 2′,5′-ADP Sepharose, fast protein liquid chromatography (FPLC)-anion exchange, and FPLC-hydrophobic interaction chromatography. The purified glutathione reductase was resolved into six isoforms by chromatofocusing. The isoform eluting with an isoelectric point of 4.9 accounted for 18% of the total activity. The five isoforms with isoelectric points between 4.1 and 4.8 accounted for 82% of the activity. Purified glutathione reductase from isolated, intact chloroplasts also resolved into six isoforms after chromatofocusing. The isoform eluting at pH 4.9 constituted a minor fraction of the total activity. By comparing the chromatofocusing profile of the seedling extract with that of the chloroplast extract, we inferred that the least acidic isoform was extraplastidic and that the five isoforms eluting from pH 4.1 to 4.8 were plastidic. Both the plastidic (five isoforms were pooled) and extraplastidic glutathione reductases had a native molecular mass of 114 kD. The plastidic glutathione reductase is a homodimer with a subunit molecular mass of 55 kD. Both glutathione reductases had optimum activity at pH 7.8. The K_m for the oxidized form of glutathione (GSSG) was 56.0 and 33.8 μm for plastidic and extraplastidic glutathione reductase, respectively, at 25°C. The K_m for NADPH was 4.8 and 4.0 μm for plastidic and extraplastidic isoforms, respectively. Antiserum raised against the plastidic glutathione reductase recognized a 55-kD polypeptide from purified antigen on western blots. In addition to the 55-kD polypeptide, another 36-kD polypeptide appeared on western blots of leaf crude extracts and the purified extraplastidic isoform. The lower molecular mass polypeptide might represent GSSG-independent enzyme activity observed on activity-staining gels of crude extracts or a protein that has an epitope similar to that in glutathione reductase. Fumigation with 75 nL L⁻¹ ozone for 4 h on 2 consecutive days had no significant effect on glutathione reductase activity in peas (Pisum sativum L.). However, immunoblotting showed a greater level of glutathione reductase protein in extracts from ozone-fumigated plants compared with that in control plants at the time when the target concentration was first reached, approximately 40 min from the start of the fumigation, and 4 h on the first day of fumigation.     

Hemocyanin-derived phenoloxidase activity in the spiny lobster Panulirus argus (Latreille, 1804)

Hemocyanin and phenoloxidase belong to the type-3 copper protein family, sharing a similar active center whereas performing different roles. In this study, we demonstrated that purified hemocyanin (450 kDa) from the spiny lobster Panulirus argus shows phenoloxidase activity in vitro after treatment with trypsin, chymotrypsin and SDS (0.1% optimal concentration), but it is not activated by sodium perchlorate or isopropanol. The optimal pHs of the SDS-activated hemocyanin were 5.5 and 7.0. Hemocyanin from spiny lobster behaves as a catecholoxidase. Kinetic characterization using dopamine, L-DOPA and catechol shows that dopamine is the most specific substrate. Catechol and dopamine produced substrate inhibition above 16 and 2 mM respectively. Mechanism-based inhibition was also evidenced for the three substrates, being less significant for L-DOPA. SDS-activated phenoloxidase activity is produced by the hexameric hemocyanin. Zymographic analysis demonstrated that incubation of native hemocyanin with trypsin and chymotrypsin, produced bands of 170 and 190 kDa respectively, with intense phenoloxidase activity. Three polypeptide chains of 77, 80 and 89 kDa of hemocyanin monomers were identified by SDS-PAGE. Monomers did not show phenoloxidase activity induced by SDS or partial proteolysis.     

Purification and characterization of a strictly specific beta-D-fucosidase from Aspergillus phoenicis.

Although beta-D-fucosidase (beta-D-fucohydrolase, EC 3.2.1.38) has been isolated from various sources, all those enzymes were associated with a high activity of beta-D-galactosidase and/or beta-D-glucosidase. We have purified a specific beta-D-fucosidase in electrophoretically homogeneous form from crude extracts of Aspergillus phoenicis by polyethyleneglycol 8000-phosphate buffer aqueous two-phase separation, and successive chromatography on DEAE-Sephadex A-50, hydroxyapatite, and Sephadex G-100 columns. The molecular weight of the enzyme was estimated to be 57,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 50,000 to 60,000 by gel filtration on Sephadex G-100. The enzyme showed optimum activity at pH 6.0 and 40 degrees C; it was stable in the pH range 5.5-6.5 and below 35 degrees C. The Km and the Vmax values for pNP-beta -D-fucoside were 2.4 mM, and 12.8 mumol.min-1.mg-1, respectively. The enzyme was strongly inhibited by sulfhydryl group reagents, p-chloromercuribenzoate, n-ethylmaleimide, and iodoacetate. It was also inhibited by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, diethyl pyrocarbonate, and N-bromosuccinimide. Thus, -SH and -COOH groups and histidyl and tryptophyl residues were essential for enzyme activity. The purified beta-D-fucosidase showed high specificity toward p-nitrophenyl-beta-D-fucoside. The enzyme was inhibited by D-fucose and D-fucono-gamma-lactone, but not by D-galactose, D-galactono-gamma-lactone, D-glucose, or D-glucono-gamma-lactone; the latter compounds are specific inhibitors of beta-D-galactosidase and beta-D-glucosidase, respectively. Thus, this enzyme is the most strictly specific beta-D-fucosidase when compared with those previously reported.     

Changes in tyrosinase specificity by ionic liquids and sodium dodecyl sulfate

Tyrosinase is a member of the type 3 copper enzyme family involved in the production of melanin in a wide range of organisms. The ability of tyrosinases to convert monophenols into diphenols has stimulated studies regarding the production of substituted catechols, important intermediates for the synthesis of pharmaceuticals, agrochemicals, polymerization inhibitors, and antioxidants. Despite its enormous potential, the use of tyrosinases for catechol synthesis has been limited due to the low monophenolase/diphenolase activity ratio. In the presence of two water miscible ionic liquids, [BMIM][BF₄] and ethylammonium nitrate, the selectivity of a tyrosinase from Bacillus megaterium (TyrBm) was altered, and the ratio of monophenolase/diphenolase activity increased by up to 5-fold. Furthermore, the addition of sodium dodecyl sulphate (SDS) at levels of 2–50 mM increased the activity of TyrBm by 2-fold towards the natural substrates l-tyrosine and l-Dopa and 15- to 20-fold towards the non-native phenol and catechol. The R209H tyrosinase variant we previously identified as having a preferential ratio of monophenolase/diphenolase activity was shown to have a 45-fold increase in activity towards phenol in the presence of SDS. We propose that the effect of SDS on the ability of tyrosinase to convert non-natural substrates is due to the interaction of surfactant molecules with residues located at the entrance to the active site, as visualized by the newly determined crystal structure of TyrBm in the presence of SDS. The effect of SDS on R209 may enable less polar substrates such as phenol and catechol, to penetrate more efficiently into the enzyme catalytic pocket.     

A 54 kDa cysteine protease purified from the crude extract of Neodiplostomum seoulense adult worms

As a preliminary study for the explanation of pathobiology of Neodiplostomum seoulense infection, a 54 kDa protease was purified from the crude extract of adult worms by sequential chromatographic methods. The crude extract was subjected to DEAE-Sepharose Fast Flow column, and protein was eluted using 25 mM Tris-HCl (pH 7.4) containing 0.05, 0.1, 0.2 and 0.4 M NaCl in stepwise elution. The 0.2 M NaCl fraction was further purified by Q-Sepharose chromatography and protein was eluted using 20 mM sodium acetate (pH 6.4) containing 0.05, 0.1, 0.2 and 0.3 M NaCl, respectively. The 0.1M NaCl fraction showed a single protein band on SDS-PAGE carried out on a 7.5-15% gradient gel. The proteolytic activities of the purified enzyme were specifically inhibited by L-trans-epoxy-succinylleucylamide (4-guanidino) butane (E-64) and iodoacetic acid. The enzyme, cysteine protease, showed the maximum proteolytic activity at pH 6.0 in 0.1 M buffer, and degraded extracellular matrix proteins such as collagen and fibronectin with different activities. It is suggested that the cysteine protease may play a role in the nutrient uptake of N. seoulense from the host intestine.     

Purification of NADH: Hypothiocyanite oxidoreductase inStreptococcus sanguis

NADH: hypothiocyanite oxidoreductase (NHOR) activity, found in some oralStreptococci,is postulated to protect these microorganisms against salivary peroxidase-produced hypothiocyanite. NHOR, however, has not been purified so far. The purification of NHOR from crude extracts ofStreptococcus sanguisNCTC 7863 strain (by ultrafiltation and anion-exchange chromatography) revealed one fraction of 125 ± 1 kDa. However, SDS–PAGE electrophoresis provided a single protein of 21.1 ± 1.2 kDa. This last discovery suggests that NHOR enzyme is a hexameric complex having six subunits.     

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.