Gewinnung und Charakterisierung von Prolidase aus,Escherichia coli B., I. Gewinnung des Enzyms

An enzyme being able to hydrolize the imido linkage at the N-terminal end of proline is isolated from E. coli B. This fact corresponds to the specifity of hydrolization of the animal prolidase. Enzyme synthesis within the cells of E. coli B is carried out independently from growth. Changed environmental factors may influence the formation rate of enzyme in a restricted way. A relatively high enzyme biosynthesis can be reached by cultivating the strain E. coli B at a temperature of 37°C as well as an initial pH-value of the medium of 7.0 in submerged culture (400–500 rpm) By variation of the medium composition enzyme synthesis does not change considerably, however, biomass yield can be increased about 100% If mechanical cell desintegration is optimized by means of ultrasonic or vibration homogenisator the cell components may be easily released with a higher proline specific activity as animal prolidase.     

Production and Purification of a New Chillproofing Enzyme and Its Properties

Chillproofing enzyme was obtained from broth cultures of Serratia marcescens B–103. This extracellular enzyme, tentatively, named the S-enzyme was highly purified from the culture supernatant by ammonium sulfate precipitation, ethanol fractionation, gel filtration on Sephadex G–200 and column chromatography on DEAE-Sephadex A–50.

The purified preparation appeared homogeneous on a ultracentrifugation with a sedimentation coefficient of 3.14 S and a molecular weight of 38,000~45,000 determined by the method of Whitaker.

The S-enzyme hydrolyzed various proteins at pH 4~6 and at low temperature hydrolyzed nitrogenous substances which may cause chill haze in beer. So the chillproofing activity of the S-enzyme may be due to its proteolytic activity.

The S-enzyme was stable at 4°C at pH 5~10.5. It was completely inactivated by heating at 60°C for 10 min, and was inactivated by Hg²⁺ and Pb²⁺ and activated by Mn²⁺, Ca²⁺. Mg²⁺ and Zn²⁺     

Thermostable glucose-tolerant glucoamylase produced by the thermophilic fungus Scytalidium thermophilum

Glucoamylase produced byScytalidium thermophilum was purified 80-fold by DEAE-cellulose, ultrafiltration and CM-cellulose chromatography. The enzyme is a glycoprotein containing 9.8% saccharide, pI of 8.3 and molar mass of 75 kDa (SDS-PAGE) or 60 kDa (Sepharose 6B). Optima of pH and temperature with starch or maltose as substrates were 5.5/70 °C and 5.5/65 °C, respectively. The enzyme was stable for 1 h at 55 °C and for about 8 d at 4 °C, either at pH 7.0 or pH 5.5. Starch, amylopectin, glycogen, amylose and maltose were the substrates preferentially hydrolyzed. The activity was activated by 1 mmol/L Mg²⁺ (27%), Zn²⁺ (21%), Ba²⁺ (8%) and Mn²⁺ (5%).K _m and {ie11-1} values for starch and maltose were 0.21 g/L, 62 U/mg protein and 3.9 g/L, 9.0 U/mg protein, respectively. Glucoamylase activity was only slightly inhibited by glucose up to a 1 mol/L concentration.     

Partial purification and characterization of a novel Mg2+- dependent ATPase present in the cytosol from human erthrocytes

Mg²⁺-ATPase activity was identified in the cytosol of human erythrocytes. A partial purification of this activity was achieved by an initial DEAE-Sephadex column chromatography, followed by gel filtration on Sephadex G-100 and then a second DEAE-Sephadex chromatography procedure. The enzyme appeared in the void volume of the Sephadex G-100 column and was retained on an Amicon XM100A ultrafiltration membrane. The molecular weight of the enzyme was estimated to be 113 000 from SDS gels. The above purification protocol yielded an enzyme with an optimal pH between 7.6 and 8.2. The enzyme activity increased linearly between 30 and 44°C. It was stable for several months at ?20°C. Magnesium was essential for activity, but the rate attainable with Mn²⁺ was at least as great as that due to Mg²⁺. No other divalent cation was able to substitute for Mg²⁺ or Mn²⁺. Neither low nor high Ca²⁺ concentrations significantly affected the enzymatic activity. Substrate specificity studies showed that ATP was the preferred substrate followed by CTP (46% of the rate produced by ATP). Hydrolysis of GTP, UTP, ITP and ADP was less than 10% of the rate seen with ATP. No phosphatase, pyrophosphatase, phosphodiesterase, hexokinase, phosphofructokinase or adenylate cyclase activity could be detected in this enzyme preparation. Calmodulin, which stimulates the (Ca²⁺ + Mg²⁺)-ATPase of the human erythrocyte membrane, failed to enhance the Mg²⁺-ATPase activity. Of considerable interest, the activity of this Mg²⁺-ATPase was enhanced approximately 5-fold by low concentrations of mercuric ion, p-hydroxymercuribenzoate and DTNB, but was much less sensitive to iodoacetamide.     

Purification and characterization of the α-glucosidase produced by thermophilic fungus <Emphasis Type="Italic">Thermoascus aurantiacus</Emphasis> CBMAI 756

An α-glucosidase enzyme produced by the fungus Thermoascus aurantiacus CBMAI 756 was purified by ultra filtration, ammonium sulphate precipitation, and chromatography using Q Sepharose, Sephacryl S-200, and Superose 12 columns. The apparent molecular mass of the enzyme was 83 kDa as determined in gel electrophoresis. Maximum activity was observed at pH 4.5 at 70°C. Enzyme showed stability stable in the pH range of 3.0–9.0 and lost 40% of its initial activity at the temperatures of 40, 50, and 60°C. In the presence of ions Na⁺, Ba²⁺, Co²⁺, Ni²⁺, Mg²⁺, Mn²⁺, Al³⁺, Zn²⁺, Ca²⁺ this enzyme maintained 90–105% of its maximum activity and was inhibited by Cr³⁺, Ag⁺, and Hg²⁺. The enzyme showed a transglycosylation property, by the release of oligosaccharides after 3 h of incubation with maltose, and specificity for short maltooligosaccharides and α-PNPG. The K_m measured for the α-glucosidase was 0.07 μM, with a V_max of 318.0 μmol/min/mg.     

Partial purification and characterization of a novel Mg- dependent ATPase present in the cytosol from human erthrocytes

Mg²⁺-ATPase activity was identified in the cytosol of human erythrocytes. A partial purification of this activity was achieved by an initial DEAE-Sephadex column chromatography, followed by gel filtration on Sephadex G-100 and then a second DEAE-Sephadex chromatography procedure. The enzyme appeared in the void volume of the Sephadex G-100 column and was retained on an Amicon XM100A ultrafiltration membrane. The molecular weight of the enzyme was estimated to be 113 000 from SDS gels. The above purification protocol yielded an enzyme with an optimal pH between 7.6 and 8.2. The enzyme activity increased linearly between 30 and 44°C. It was stable for several months at −20°C. Magnesium was essential for activity, but the rate attainable with Mn²⁺ was at least as great as that due to Mg²⁺. No other divalent cation was able to substitute for Mg²⁺ or Mn²⁺. Neither low nor high Ca²⁺ concentrations significantly affected the enzymatic activity. Substrate specificity studies showed that ATP was the preferred substrate followed by CTP (46% of the rate produced by ATP). Hydrolysis of GTP, UTP, ITP and ADP was less than 10% of the rate seen with ATP. No phosphatase, pyrophosphatase, phosphodiesterase, hexokinase, phosphofructokinase or adenylate cyclase activity could be detected in this enzyme preparation. Calmodulin, which stimulates the (Ca²⁺ + Mg²⁺)-ATPase of the human erythrocyte membrane, failed to enhance the Mg²⁺-ATPase activity. Of considerable interest, the activity of this Mg²⁺-ATPase was enhanced approximately 5-fold by low concentrations of mercuric ion, p-hydroxymercuribenzoate and DTNB, but was much less sensitive to iodoacetamide.     

Polynucleotide Phosphorylase from Acromobacter sp. KR 170-4

Eighty-five strains of bacteria were screened for selection of microorganisms suitable for industrial production of polynucleotides. Among these bacteria, Achromobacter sp. KR 170-4 (ATCC 21942) was found to be rich in polynucleotide Phosphorylase (PNPase) in its “salt-shockate” as compared with the other strains tested. PNPase was purified about 50-fold from the “salt-shockate” of Achromobacter sp. KR 170-4, and properties of the enzyme were elucidated. Optimal pH for reaction was 10.1. Stable pH range at 37°C was between pH 6.5 and 10.5. Optimal temperatures were 46°C for polymerization of ADP or IDP, and 43°C for CDP or UDP. The enzyme was stable below 55°C at pH 9.2. The enzyme required Mn²⁺ rather than Mg²⁺ unlike the other PNPases reported. Optimal concentration of Mn²⁺ was 6 mM.     

Comparison of the effect of calcium(II) and manganese(II) ions on trypsin autolysis

The effect of Mn²⁺ and Ca²⁺ ions on the rate of trypsin autolysis was studied at pH 7.0 and at 34.4-60.2°C. For comparison, the kinetic constants of esterolytic activity of trypsin in the presence of the metal ion were determined at pH 7.4 and at 36° and 40°C. There was no significant difference in the rate of autolysis between Mn²⁺ and Ca²⁺ in the temperature range 34-47°C, but at 56.8° and 60.2° autolysis was slightly more rapid in the presence of Mn²⁺. The Mn²⁺ or Ca²⁺ ion bound to trypsin is supposed to control the conformation and thereby the stability and the activity of the enzyme. This indirect effect of Mn²⁺ and Ca²⁺ is discussed on a structural basis of the enzyme molecule.     

Glutathione Synthetase of Aspergillus niger: Structural Properties of the Enzyme in Prokaryotes and Eukaryotes

Glutathione synthetase isolated from a mold, Aspergillus niger, had a molecular weight of 110,000 and consisted of two apparently identical subunits, each with a molecular weight of 55,000. The enzyme was most active at pH 8.5. It specifically utilized glycine and ATP, and required Mg^{2 +} or Mn^{2 +} for its catalytic function. A comparison of glutathione synthetases from various sources indicated that the enzyme of eukaryotes (mammals, molds and yeasts) differ from those of prokaryotes {Escherichia coli B and Proteus mirabilis) in molecular structure, although the enzymes from both types of organisms contain an active site thiol and catalyze the same reaction.     

Spectroscopic identification of a dinuclear metal centre in manganese(II)-activated aminopeptidase P from Escherichia coli: implications for human prolidase

Electron paramagnetic resonance (EPR) spectra and X-ray absorption (EXAFS and XANES) data have been recorded for the manganese enzyme aminopeptidase P (AMPP, PepP protein) from Escherichia coli. The biological function of the protein, a tetramer of 50-kDa subunits, is the hydrolysis of N-terminal Xaa-Pro peptide bonds. Activity assays confirm that the enzyme is activated by treatment with Mn²⁺. The EPR spectrum of Mn²⁺–activated AMPP at liquid-He temperature is characteristic of an exchange-coupled dinuclear Mn(II) site, the Mn-Mn separation calculated from the zero-field splitting D of the quintet state being 3.5?(±0.1)?Å. In the X-ray absorption spectrum of Mn²⁺–activated AMPP at the Mn K edge, the near-edge features are consistent with octahedrally coordinated Mn atoms in oxidation state +2. EXAFS data, limited to k≤12?Å^–1 by traces of Fe in the protein, are consistent with a single coordination shell occupied predominantly by O donor atoms at an average Mn-ligand distance of 2.15?Å, but the possibility of a mixture of O and N donor atoms is not excluded. The Mn-Mn interaction at 3.5?Å is not detected in the EXAFS, probably due to destructive interference from light outer-shell atoms. The biological function, amino acid sequence and metal-ion dependence of E. coli AMPP are closely related to those of human prolidase, an enzyme that specifically cleaves Xaa-Pro dipeptides. Mutations that lead to human prolidase deficiency and clinical symptoms have been identified. Several known inhibitors of prolidase also inhibit AMPP. When these inhibitors are added to Mn²⁺–activated AMPP, the EPR spectrum and EXAFS remain unchanged. It can be inferred that the inhibitors either do not bind directly to the Mn centres, or substitute for existing Mn ligands without a significant change in donor atoms or coordination geometry. The conclusions from the spectroscopic measurements on AMPP have been verified by, and complement, a recent crystal structure analysis.     

Distribution de la Phosphonates dans le Sang et le Sperme de Boeuf

A new assay for aminopeptidase P was established by coupling with proline iminopeptidase using Gly-Pro-chromogen (e.g. Gly-Pro-β-naphthylamide, Gly-Pro-p-nitroanilide, or .Gly-Pro-4-methyl coumarin amide) as the substrate. With each substrate, a linear relationship was established between the enzyme amounts and color development or fluorescence due to the chromogen released. This assay method did not suffer from interference by materials in culture broth. By using this assay method, aminopeptidase P was partially purified from Escherichia coli HB101 by chromatographies on DEAE-Sephadex and high performance liquid chromatography (HPLC). On the chromatogram with a DEAE-Sephadex column, two peaks of aminopeptidase P were observed and were named APP-I and APP-II. APP-I was further purified by HPLC using DEAE-5PW and Phenyl-5PW columns. Optimum pHs of APP-I and APP-II were 8.0 and 9.0, respectively. In contrast to APP-I which was stable around pH 10, APP-II was stable at pH 8 to 9. After incubation for 30 min at pH 8.0, fifty percent of the remaining activity of APP-I and APP-II were observed at 60°C and 50°C. APP-I and APP-II were activated 3-fold by the addition of 5 and 30 μm Mn²⁺. They were inhibited by EDTA, and reactivated by adding Mn^{2 +}. The molecular weights of APP-I and APP-II were 350,000 and 210,000, respectively. Each enzymes released the amino terminal amino acid when proline is at the penultimate position. The velocity of hydrolysis by the enzymes was not significantly different for most X-Pro bonds (X = amino acid) of peptides except for Pro-Pro bond. APP-II hydrolyzed penta-(Pro-Pro-Gly) at a much higher rate than APP-I, suggesting the aminopeptidase P reported by Yaron and Mlynar (BBRC, 32, 658 (1968)) to be APP-II.     

A New Natural Quinone, 2-Methyl-3-II,III,VIII-hexahydromultiprenyl9-1,4-naphthoquinone

A new aryl-peptidyl amidase has been isolated from a Lactobacillus casei homogenate. Its ribosomal localization was shown by fractionation and its general properties studied after purification on Sepharose 6B and DEAE-Sephacel. The enzyme requires 1 mM Mg²⁺ for stability, while Zn²⁺, Mn²⁺, Co²⁺ and Ca²⁺ result in only partial stability. No inhibitory effects were noted after treatment with phenylmethylsulfonylfluoride or EDTA. Enzymatic activity was totally inhibited by 5mM p-hydroxymercuribenzoate; activity was restored by dithiothreitol. The only substrates hydrolyzed by this enzyme were the succinyl-L-phenylalanine-p-nitroanilide type, with a pH optimum between 6 and 7 and a Michaelis constant of 0.76 mM. No hydrolysis could be detected using proteins, peptides, amides or esterase substrates. This enzyme would thus not be an endopeptidase (E.C. 3.4.21), but would to rather be considered as belonging to the group of amidases (E.C. 3.5.1)     

Isolation and characterization of β-galactosidase fromLactobacillus crispatus

β-Galactosidase was isolated from the cell-free extracts ofLactobacillus crispatus strain ATCC 33820 and the effects of temperature, pH, sugars and monovalent and divalent cations on the activity of the enzyme were examined.L. crispatus produced the maximum amount of enzyme when grown in MRS medium containing galactose (as carbon source) at 37°C and pH 6.5 for 2 d, addition of glucose repressing enzyme production. Addition of lactose to the growth medium containing galactose inhibited the enzyme synthesis. The enzyme was active between 20 and 60°C and in the pH range of 4–9. However, the optimum enzyme activity was at 45°C and pH 6.5. The enzyme was stable up to 45°C when incubated at various temperatures for 15 min at pH 6.5. When the enzyme was exposed to various pH values at 45°C for 1 h, it retained the original activity over the pH range of 6.0–7.0. Presence of divalent cations, such as Fe²⁺ and Mn²⁺, in the reaction mixture increased enzyme activity, whereas Zn²⁺ was inhibitory. TheK _m was 1.16 mmol/L for 2-nitrophenyl-β-d-galactopyranose and 14.2 mmol/L for lactose.     

Purification and Properties of Phospholipase D from Actinomadura sp. Strain No. 362

An extracellular phospholipase D from Actinomadura sp. Strain No. 362 was purified about 430-fold from the culture filtrate. The purified enzyme preparation was judged to be homogeneous on polyacrylamide gel electrophoresis. The molecular weight and isoelectric point of the enzyme were estimated to be about 50,000—60,000 and 6.4, respectively. The enzyme was most active at pH 5.5 and 50°C in the presence of Triton X-100, but showed the highest activity at pH 7.0 and 60 — 70°C in its absence. The enzyme was stable up to 30°C at pH 7.2 and also stable in the pH range of 4.0 to 8.0 on 2 hr incubation at 25°C. With regard to substrate specificity, this enzyme hydrolysed lecithin best among the phospholipids tested. It was activated by Fe^{3 +}, Al³⁺, Mn^{2 +}, Ca^{2 +}, diethyl ether, sodium deoxycholate and Triton X-100, but was inhibited by cetyl pyridinium chloride and dodecylsulfate.     

Isolation and characterization of a manganese-stimulated exonuclease fromBacillus subtilis

A manganese-stimulated exonuclease was purified from culture fluids ofBacillus subtilis 168 using ammonium sulfate fractionation, SephadexG-150 gel filtration, and DEAE-Sephadex ion-exchange chromatography. This extracellular nuclease was found to attain maximal activity in the presence of 5 mM Mn²⁺. Little or no activity was demonstrated in the presence of Fe²⁺, Mg²⁺, Zn²⁺, Cu²⁺, or Ni²⁺, but the nuclease was somewhat active with Ca²⁺. The nuclease exhibited a broad pH range, with maximum activity at pH 8.5. A molecular weight of 214,000 was calculated for the protein using Sephacryl S-300 columm chromatography. Incubation of the enzyme with the closed circular DNA of plasmid pUB110 indicated that the nuclease is strictly, exonucleolytic in nature.     

Preparation of Zeaxanthin from Berries of Boxthorn,Lycium chinensis

Purification and properties of a new alkaline protease of rat skeletal muscle have been reported. The purification procedure of the enzyme is as follows: skeletal muscle tissue was extracted successively with Hasselbach-Schneider solution, 5 m urea solution and 2% sodium deoxycholate solution. After then, the enzyme was extracted from the residue with 1.1 m potassium iodide solution. This enzyme solution was treated with n-butanol, and dialyzed against water. The enzyme precipitated during dialysis was collected and dissolved in 1.1 m potassium iodide solution. The enzyme solution was fractionated with acetone, and chromatographed on Sephadex G-200. The final preparation showed over 20,000 times of purity.

The optimum pH range of the enzyme activity is 9.5~10.5, and the maximum reaction rate occurs at 47~57°C. The enzyme is stable below 47°C at pH 7.3. At 37°C, the enzyme is stable during 30 min at least, in the pH range of 5.5~10.0. Below pH 5.0, it is relatively labile. Hg²⁺, Ca²⁺, Mg²⁺, Mn²⁺, Co²⁺, and Zn²⁺ scarcely affect the enzyme activity at the concentration of 1 mm. Ethylenediaminetetraacetate shows little effect on the activity at the concentration of 10 mm, and iodoacetamide, 2,4-dinitrophenol, p-chloromercuribenzoate show the similar effect at the concentration of 1 mm. Diisopropyl-flurophosphate inhibits the enzyme activity. From the results obtained, this enzyme is presumed to be responsible for the activity of autolytic breakdown of rat skeletal muscle proteins in the alkaline pH range.     

Regulation,purification and partial characterization of glutamine synthetase fromStreptomyces aureofaciens

The activity of glutamine synthetase (GS) fromStreptomyces aureofaciens was regulated by the availability of the nitrogen source. Rich nitrogen sources repressed GS synthesis and increased GS adenylylation. The enzyme was purified 270-fold to virtual homogeneity with 37% recovery. The molar mass of the native enzyme and its subunits was determined to be 620 and 55 kDa, respectively, indicating that GS is composed of 12 identical subunits. The enzyme has a hexagonal-bilayered structure as observed by electron microscopy. The isoelectric point of the purified GS was at pH 4.2. The enzyme was stable for 1 h at 50°C but lost activity rapidly when incubated at 65 and 70°C. Mg²⁺ supported relative synthetic activity of 100 and 72%, respectively, with the corresponding pH optima of 7.3 and 7.0. Mn²⁺ ions activated transferase activity at a pH optimum of 7.0. The temperature optimum for all GS activities was 50°C. Intermediates of the citric acid cycle exerted insignificant effects on the synthetic activities. There was no SH-group essential for the GS activity.     

Production,purification and characterization of <Emphasis Type="Italic">Bacillus</Emphasis> sp. GRE7 xylanase and its application in eucalyptus Kraft pulp biobleaching

Production of extracellular xylanase from Bacillus sp. GRE7 using a bench-top bioreactor and solid-state fermentation (SSF) was attempted. SSF using wheat bran as substrate and submerged cultivation using oat-spelt xylan as substrate resulted in an enzyme productivity of 3,950 IU g⁻¹ bran and 180 IU ml⁻¹, respectively. The purified enzyme had an apparent molecular weight of 42 kDa and showed optimum activity at 70°C and pH 7. The enzyme was stable at 60–80°C at pH 7 and pH 5–11 at 37°C. Metal ions Mn²⁺ and Co²⁺ increased activity by twofold, while Cu²⁺ and Fe²⁺ reduced activity by fivefold as compared to the control. At 60°C and pH 6, the K _m for oat-spelt xylan was 2.23 mg ml⁻¹ and V _max was 296.8 IU mg⁻¹ protein. In the enzymatic prebleaching of eucalyptus Kraft pulp, the release of chromophores, formation of reducing sugars and brightness was higher while the Kappa number was lower than the control with increased enzyme dosage at 30% reduction of the original chlorine dioxide usage. The thermostability, alkali-tolerance, negligible presence of cellulolytic activity, ability to improve brightness and capacity to reduce chlorine dioxide usage demonstrates the high potential of the enzyme for application in the biobleaching of Kraft pulp.     

Purification,characterization and antitumor activity of l-asparaginase isolated from Pseudomonas stutzeri MB-405

An l-asparaginase produced by Pseudomonas stutzeri MB-405 was isolated and characterized. After initial ammonium sulfate fractionation, the enzyme was purified by consecutive column chromatography on Sephadex G-100, Ca-hydroxylapatite, and DEAE-Sephadex A-50. The 665.5-fold purified enzyme thus obtained has the specific activity of 732.3 units mg protein^-1 with an overall recovery of 27.2%. The apparent M _r of the enzyme under nondenaturing and denaturing conditions was 34 kDa and 33 kDa respectively, and the isoelectric point was 6.38±0.02. It displayed optimum activity at pH 9.0 and 37°C. The enzyme was very specific for l-asparagine and did not hydrolyze L-glutaminate. The K _m of the l-asparaginase was found to be 1.45×10^-4 m towards l-asparagine and was competitively inhibited by 5-diazo-4-oxo-l-norvaline (DONV) with a K _i of 0.03mm. Metal ions such as Mn²⁺, Zn²⁺, Hg²⁺, Fe³⁺, Ni²⁺, and Cd²⁺ potentially inhibited the enzyme activity. The activity was enhanced in the presence of thiol-protecting reagents such as DTT, 2-ME, and glutathione (reduced), but inhibited by PCMB and iodoacetamide. The tumor inhibition study with Dalton's lymphoma tumor cells in vivo indicated that this enzyme possesses antitumor properties.     

Kinetic and redox properties of MnP II, a major manganese peroxidase isoenzyme from Panus tigrinus CBS 577.79

A manganese peroxidase (MnP) isoenzyme from Panus tigrinus CBS 577.79 was produced in a benchtop stirred-tank reactor and purified to apparent homogeneity. The purification scheme involving ultrafiltration, affinity chromatography on concanavalin–A Sepharose, and gel filtration led to a purified MnP, termed “MnP II,” with a specific activity of 288 IU mg⁻¹ protein and a final yield of 22%. The enzyme turned out to be a monomeric protein with molecular mass of 50.5 kDa, pI of 4.07, and an extent of N-glycosylation of about 5.3% of the high-mannose type. The temperature and pH optima for the formation of malonate manganic chelates were 45 °C and 5.5, respectively. MnP II proved to be poorly thermostable at 50 and 60 °C, with half-lives of 11 min and 105 s, respectively. K _m values for H₂O₂ and Mn²⁺ were 16 and 124 μM, respectively. Although MnP II was able to oxidize veratryl alcohol and to catalyze the Mn²⁺-independent oxidation of several phenols, it cannot be assigned to the versatile peroxidase family. As opposed to versatile peroxidase oxidation, veratryl alcohol oxidation required the simultaneous presence of H₂O₂ and Mn²⁺; in addition, low turnover numbers and K _m values higher than 300 μM characterized the Mn²⁺-independent oxidation of substituted phenols. Kinetic properties and the substrate specificity of the enzyme markedly differed from those reported for MnP isoenzymes produced by the reference strain P. tigrinus 8/18. To our knowledge, this study reports for the first time a thorough electrochemical characterization of a MnP from this fungus.