Purification and Identification of a Novel Leucine Aminopeptidase from <Emphasis Type="Italic">Bacillus thuringiensis israelensis</Emphasis>

A novel leucine aminopeptidase was purified from a Bacillus thuringiensis israelensis (Bti) culture. The purification stages included heating the concentrated supernatant to 65°C for 90 min, anion-exchange chromatography by DEAE cellulose, and hydrophobic chromatography by phenyl Sepharose. The specific activity of leucine aminopeptidase after the hydrophobic chromatography increased by 215.5-fold and the yield was 16%. The molecular weight of the active enzyme was 59 kDa. Mass spectrometry analysis of the 59-kDa leucine aminopeptidase revealed that this protein has at least 41% homology with the cytosol leucine aminopeptidase produced by Bacillus cereus. Maximal leucine aminopeptidase activity occurred at 65°C, pH 10 toward leucine as the amino acid terminus. The enzyme was strongly inhibited by bestatin, dithiothreitol, and 1,10-phenanthroline, indicating that the enzyme might be considered as a metallo-aminopeptidase that has disulfide bonds at the catalytic site or at a region that influences its configuration. Examination of the purified leucine aminopeptidase’s effect on the activation of the protoxin Cyt1Aa from Bti revealed that when it acts synergistically with Bti endogenous proteases, it has only a minor role in the processing of Cyt1Aa into an active toxin.     

An Aneurinibacillus sp. strain AM-1 produces a proline-specific aminopeptidase useful for collagen degradation

AIMS: We have been for a species of thermophilic bacteria that can effectively decompose collagen and collagen peptides that tend to be hard-to-degrade proteins because of their high content of proline residues. This study focused upon the enzymatic degradation of prolyl peptides by thermophilic bacteria. METHODS AND RESULTS: A strain, AM-1, producing a proline-specific aminopeptidase was isolated using a medium containing gelatin that was taken from soil samples collected at Arima Hot Spring located near Kobe, Japan. The strain showed the strongest level of hydrolysing activity toward prolyl-p-nitroanilide, and the activity proved to be thermostable. Phylogenetic analysis based on 16S rDNA sequences revealed that the isolated strain AM-1 was closest to Aneurinibacillus thermoaerophilus DSM10154T in its characteristics. Analysis of the purified proline-specific aminopeptidase suggested that the enzyme is an aminopeptidase containing metal that includes important disulphide bond(s). The strain AM-1 aminopeptidase has more similarities with leucyl aminopeptidases, but its activity level differs greatly with prolyl peptides. CONCLUSIONS: The proline-specific aminopeptidase from strain AM-1 is the first from the genus Aneurinibacillus and may be a new type of aminopeptidase for hydrolysing prolyl peptide. This enzyme also contributed to the degradation of collagen when used in combination with another collagenolytic protease. SIGNIFICANCE AND IMPACT OF THE STUDY: The proline-specific aminopeptidase obtained from strain AM-1 may be used in the treatment of wastewater containing collagen that is encountered in the meat industries, and for decreasing bitter peptides in milk products.     

Enzymatic properties of the glycine d-alanine aminopeptidase of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> and its activity profiles in liquid-cultured mycelia and solid-state rice culture (rice koji)

The gdaA gene encoding S12 family glycine–d-alanine aminopeptidase (GdaA) was found in the industrial fungus Aspergillus oryzae. GdaA shares 43% amino acid sequence identity with the d-aminopeptidase of the Gram-negative bacterium Ochrobactrum anthropi. GdaA purified from an A. oryzae gdaA-overexpressing strain exhibited high d-stereospecificity and efficiently released N-terminal glycine and d-alanine of substrates in a highly specific manner. The optimum pH and temperature were 8 to 9 and 40°C, respectively. This enzyme was stable under alkaline conditions at pH 8 to 11 and relatively resistant to acidic conditions until pH 5.0. The chelating reagent EDTA, serine protease inhibitors such as AEBSF, benzamidine, TPCK, and TLCK, and the thiol enzyme inhibitor PCMB inhibited the enzyme. The aminopeptidase inhibitor bestatin did not affect the activity. GdaA was largely responsible for intracellular glycine and d-alanine aminopeptidase activities in A. oryzae during stationary-phase growth in liquid media. In addition, the activity increased in response to the depletion of nitrogen or carbon sources in the growth media, although the GdaA-independent glycine aminopeptidase activity highly increased simultaneously. Aminopeptidases of A. oryzae attract attention because the enzymatic release of a variety of amino acids and peptides is important for the enhancement of the palatability of fermented foods. GdaA activity was found in extracts of a solid-state rice culture of A. oryzae (rice koji), which is widely used as a starter culture for Japanese traditional fermented foods, and was largely responsible for the glycine and d-alanine aminopeptidase activity detected at a pH range of 6 to 9.     

Novel prolyl tri/tetra-peptidyl aminopeptidase from Streptomyces mobaraensis: substrate specificity and enzyme gene cloning

The prolyl peptidase that removes the tetra-peptide of pro-transglutaminase was purified from Streptomyces mobaraensis mycelia. The substrate specificity of the enzyme using synthetic peptide substrates showed proline-specific activity with not only tripeptidyl peptidase activity, but also tetrapeptidyl peptidase activity. However, the enzyme had no other exo- and endo-activities. This substrate specificity is different from proline specific peptidases so far reported. The enzyme gene was cloned, based on the direct N-terminal amino acid sequence of the purified enzyme, and the entire nucleotide sequence of the coding region was determined. The deduced amino acid sequence revealed an N-terminal signal peptide sequence (33 amino acids) followed by the mature protein comprising 444 amino acid residues. This enzyme shows no remarkable homology with enzymes belonging to the prolyl oligopeptidase family, but has about 65% identity with three tripeptidyl peptidases from Streptomyces lividans, Streptomyces coelicolor, and Streptomyces avermitilis. Based on its substrate specificity, a new name, "prolyl tri/tetra-peptidyl aminopeptidase," is proposed for the enzyme.     

Functional characterization of an Arabidopsis prolyl aminopeptidase AtPAP1 in response to salt and drought stresses

It has been over 50 years since the first prolyl aminopeptidase gene was identified in Escherichia coli (EC 3.4.11.5). However, up to now, few prolyl aminopeptidases have been reported to regulate osmotic stress tolerance, especially in plant. In this study, we focused on characterization of the biological functions of the Arabidopsis prolyl aminopeptidase AtPAP1 (At2g14260), which positively regulated plant tolerance to salt and drought stresses. Protein sequence alignment revealed that AtPAP1 was evolutionarily conserved among different plant species, and the smaller molecular weight and phylogenetic tree indicated that AtPAP1 belonged to the S33.001 subfamily. By using quantitative real-time PCR assays, we demonstrated that expression of the AtPAP1 gene was rapidly induced by salt and drought stresses. We also found that knockout of the AtPAP1 gene decreased, while AtPAP1 overexpression enhanced plant tolerance to salt and drought stresses. Measurements of the proline contents and the prolyl aminopeptidase activity suggested that the transgenic plants accumulated more free proline and exhibited higher prolyl aminopeptidase activity than the wild type or knockout plants under control conditions, as well as salt and drought stresses. Furthermore, through the GUS activity analysis, we also demonstrated that the AtPAP1 promoter is stress inducible and tissue specific. The AtPAP1-GFP fusion protein was found to localize in the cytoplasm of the onion epidermal cells. In conclusion, we showed that the Arabidopsis AtPAP1 gene could positively regulate plant tolerance to salt and drought stress, maybe by acting as a prolyl aminopeptidase and thereby increasing the concentration of free proline in plant cells.     

Expression and Purification of a Recombinant “Small” Sialidase fromClostridium perfringensA99

A 1.4-kb gene encoding the “small” sialidase isoenzyme ofClostridium perfringensA99, including its own promoter, was previously cloned in and expressed byEscherichia coliJM 101. Since all attempts to purify this enzyme to homogeneity were unsuccessful, a new strategy was developed. The structural gene was amplified by means of a PCR technique and inserted into the plasmid vector pQE-10, transferring a six-histidine affinity tag (His₆) to the N-terminus of the protein. In order to minimize proteolytic degradation of the sialidase protein, the gene was subcloned into theEscherichia colistrain BL21(DE3)pLys S with reduced protease activity. The sialidase production was increased about 2.5-fold when compared with that of the original clone. The enzyme, released by lysozyme treatment of the bacterial cells, was purified by metal chelate chromatography on Ni–nitrilo-triacetic acid agarose to apparent homogeneity in SDS–PAGE. The 42-kDa protein was enriched 62-fold with a yield of 82% and a specific activity of 280 U mg⁻¹. A total amount of 1 mg sialidase was obtained from 1 liter of bacterial culture. For future studies, including crystallization experiments, the histidine affinity tag was removed from the sialidase enzyme by aminopeptidase K. The sialidase was then separated from aminopeptidase K by ion-exchange chromatography, resulting in an overall yield of 83% and a specific activity of 305 U mg⁻¹using 4-methylumbelliferyl-α- -N-acetylneuraminic acid under standard conditions. The two forms (with or without the histidine tag) of sialidase exhibited similar kinetic properties when compared to the wild-type enzyme.     

An activated by cobalt alkaline aminopeptidase from <Emphasis Type="Italic">Bacillus mycoides</Emphasis>

An intracellular arginine—specific aminopeptidase synthesized by Bacillus mycoides was purified and characterized. The purification procedure for studied aminopeptidase consisted of ammonium sulphate precipitation and three chromatographic steps: anion exchange chromatography and gel permeation chromatography. A molecular weight of ∼50 kDa was estimated for the aminopeptidase by gel permeation chromatography and SDS-PAGE. The optimal activity of the enzyme on arginyl-β-naphthylamide as a substrate was at 37°C and pH 9.0. The enzyme showed maximum specificity for basic amino acids: such as Arg and Lys but was also able to hydrolyze aromatic amino acids: Trp, Tyr, and Phe. Co²⁺ ions activated the enzyme, while Zn²⁺, Cu²⁺, Hg²⁺ and Mn²⁺ inhibited it. The enzyme is a metalloaminopeptidase whose activity is inhibited by typical metalloaminopeptidase inhibitors: EDTA and 1,10-phenanthroline. Analysis of fragments of the amino acid sequence of the purified enzyme demonstrated high similarity to Amp S of Bacillus cereus and APII of B. thuringensis.     

Structural and immunological evidence for the identity of prolyl aminopeptidase with leucyl aminopeptidase

Prolyl aminopeptidase (EC 3.4.11.5) has been assumed to be a unique enzyme catalyzing specifically the removal of unsubstituted NH2-terminal L-prolyl residues from various peptides and to be distinct from leucyl aminopeptidase (EC 3.4.11.1). In the present study, prolyl aminopeptidases were purified to apparent homogeneity from pig small intestine mucosa and human liver and their NH2-terminal amino acid sequences were determined together with that of pig kidney leucyl aminopeptidase. The NH2-terminal 24-residue sequence of pig intestinal prolyl aminopeptidase was shown to be identical with that of pig kidney leucyl aminopeptidase. The NH2-terminal sequence of human liver prolyl aminopeptidase was also shown to be very similar to that of pig kidney leucyl aminopeptidase. Further, pig intestinal prolyl aminopeptidase and pig kidney leucyl aminopeptidase were immunologically indistinguishable. These lines of evidence strongly suggest that prolyl aminopeptidase is identical with leucyl aminopeptidase.     

Characterization by molecular cloning and sequencing of the gene encoding an aminopeptidase from Listeria monocytogenes

The pepC gene of Listeria monocytogenes encodes aminopeptidase C that is predicted to share 72% amino acid sequence similarity and 53% sequence identity with the cysteine aminopeptidase PepC from Lactococcus lactis. The gene product also shows strong similarity to aminopeptidase C from Streptococcus thermophilus and Lactobacillus helveticus, and to a cysteine proteinase/bleomycin hydrolase from Saccharomyces cerevisiae. The enzyme from L. monocytogenes displayed broad N-terminal hydrolytic activity, with a similar substrate specificity to its lactic acid bacterial counterpart. The inhibition spectrum shows a great deal of similarity with enzymes from the family of lactic acid bacteria. In addition, one of the clones studied contained DNA sequences that could encode a regulatory protein of the deoR helix-turn-helix DNA binding protein family. The organization of the locus, designated pep, is presented along with the characterization of the gene products of the pep locus.     

Characterisation of the <Emphasis Type="Italic">Aspergillus niger dapB</Emphasis> gene,which encodes a novel fungal type IV dipeptidyl aminopeptidase

We have cloned the Aspergillus niger dapB gene. Analysis of its nucleotide sequence and the corresponding protein sequence indicates that the gene encodes a type IV dipeptidyl aminopeptidase (DPP IV). Based upon its deduced sequence we predict the presence of a transmembrane domain in the protein. Furthermore, dapB-overexpressing transformants display an increase in intracellular DPP IV activity. This is the first reported characterisation of a dipeptidyl aminopeptidase with a transmembrane domain from a filamentous fungus. Using the dapB sequence as a query, we were able to identify 14 DPP IV-encoding genes, and 12 additional DPPIV proteases in public genomic databases. Phylogenetic analysis reveals that in yeasts there are two clades of genes that encode DPP IV proteases with a transmembrane domain. In this study we demonstrate that, as in yeasts, two classes of DPP IV-encoding genes exist in filamentous fungi. However, only one of these codes for DPP IV proteases with a transmembrane domain. The second type present in filamentous fungi encodes extracellular DPP IV proteases. The dapB gene belongs to the first cluster. We propose that DapB plays a role in the proteolytic maturation of enzymes produced by A. niger.Electronic Supplementary Material Supplementary material is available for this article at     

Location of Peptidases Outside and Inside the Membrane of Streptococcus cremoris

Peptidase activity determinations involving native cells of Streptococcus cremoris and completely disrupted cell preparations, as well as experiments concerned with peptidase activity distribution among cell fractions obtained by a damage-restrictive removal of the cell wall and release of intracellular material, suggest the presence of peptidases with distinguishable locations. Alanyl, leucyl, and prolyl aminopeptidase activities are most likely located in the cell wall-membrane interface, showing no detectable association with the membrane. Lysyl aminopeptidase is present not only in this location, but also as an intracellular enzyme. Endopeptidase activity and glutamate aminopeptidase activity appear to be weakly associated with the membrane. The locations of these two peptidase activities, unlike those of the former aminopeptidase activities, impose a restriction on their expression. Results of experiments concerned with permeabilization of the membrane and findings regarding an effect of the local environment of the enzymes on their pH activity profiles are evaluated and considered as being indicative of the proposed location. The possible implications of these findings with respect to protein utilization during growth of the organism in milk are discussed.     

A thermostable leucine aminopeptidase from<Emphasis Type="Italic"> Bacillus kaustophilus</Emphasis> CCRC 11223

Two degenerate primers established from the consensus sequences of bacterial leucine aminopeptidases (LAP) were used to amplify a 360-bp gene fragment from the chromosomal DNA of thermophilic Bacillus kaustophilus CCRC 11223 and the amplified fragment was successfully used as a probe to clone a leucine aminopeptidase (lap) gene from a genomic library of the strain. The gene consists of an open reading frame (ORF) of 1,494 bp and encodes a protein of 497 amino acid residues with a calculated molecular mass of 53.7 kDa. The complete amino acid sequence of the cloned enzyme showed greater than 30% identity with prokaryotic and eukaryotic LAPs. Phylogenetic analysis showed that B. kaustophilus LAP is closely related to the enzyme from Bacillus subtilis and is grouped with the M17 family. His₆-tagged LAP was generated in Escherichia coli by cloning the coding region into pQE-30 and the recombinant enzyme was purified by nickel-chelate chromatography. The pH and temperature optima for the purified enzyme were 8 and 65°C, respectively, and 50% of its activity remained after incubation at 60°C for 32 min. The enzyme preferentially hydrolyzed l-leucine-p-nitroanilide (l-Leu-p-NA) followed by Cys derivative.Communicated by G. Antranikian     

Efficient production of active Vibrio proteolyticus aminopeptidase in Escherichia coli by co-expression with engineered vibriolysin

The Vibrio proteolyticus aminopeptidase is synthesized as a preproprotein and then converted into an active enzyme by cleavage of the N-terminal propeptide. In recombinant Escherichia coli, however, the aminopeptidase is not processed correctly and the less-active form that has the N-terminal propeptide accumulates in the culture medium. Recently, we isolated a novel vibriolysin that was expressed as an active form in E. coli by random mutagenesis; this enzyme shows potential as a candidate enzyme for the processing of aminopeptidase. The E. coli cells were engineered to co-express the novel vibriolysin along with aminopeptidase. Co-expression of vibriolysin resulted in an approximately 13-fold increase in aminopeptidase activity, and a further increase was observed in the form lacking its C-terminal propeptide. The active aminopeptidase was purified from the culture supernatant including the recombinant vibriolysin by heat treatment and ion exchange and hydroxyapatite chromatography with high purity and 35% recovery rate. This purified aminopeptidase effectively converted methionyl-human growth hormone (Met-hGH) to hGH. Thus, this co-expression system provides an efficient method for producing active recombinant V. proteolyticus aminopeptidase.     

Possible involvement of aminopeptidase, an ecto-enzyme, in the inactivation of bradykinin by intact neutrophils

The subcellular localization of the bradykinin-inactivating activity was studied using guinea-pig neutrophils and the following results were obtained. The bradykinin-inactivating activities were found to be present in the cytosol and membrane fractions but not in the granular and nuclear fractions. The bradykinin-inactivating activity of the cytosol fraction was inhibited by N-carbobenzoxy-Gly-Pro, an inhibitor of prolyl endopeptidase, whereas that of the membrane fraction was inhibited by bestatin, an inhibitor of aminopeptidase. Prolyl endopeptidase and aminopeptidase activities were located predominantly in the cytosol and membrane fractions, respectively, and their activities were inhibited by their respective inhibitors. Prolyl endopeptidase and aminopeptidase activities measured with synthetic substrates were competitively inhibited by bradykinin, suggesting that bradykinin is a possible substrate for prolyl endopeptidase and aminopeptidase. Intact neutrophils inactivated bradykinin rapidly. However, when neutrophils were modified chemically by diazotized sulfanilic acid, a poorly permeant reagent which inactivates ecto-enzymes selectively, both the bradykinin-inactivating activity and aminopeptidase activity of neutrophils decreased significantly without any inhibition of cytosol prolyl endopeptidase. The possibility that aminopeptidase, an ecto-enzyme, would be responsible for the inactivation of bradykinin by intact neutrophils was deduced from the results above, although both cytosol prolyl endopeptidase and membrane aminopeptidase could inactivate bradykinin.     

Regulation of Nicotine Production by Auxins in Tobacco Cultured Cells in vitro

A thermostable aminopeptidase, called aminopeptidase T, from the extract of Thermus aquaticus YT-1 was purified and characterized. The enzyme had a dimeric structure, its relative molecular mass being 108,000 by gel filtration, and 48,000 by SDS-PAGE. The optimum pH of the enzyme activity was in the range of 8.5 to 9.0. The enzyme was significantly thermostable as it still retained 60% of its original activity even after heat treatment for 20 hr at 80°C. The enzyme activity was inhibited by metal-chelating agents. The enzyme had a low substrate specificity.     

Optimized expression of prolyl aminopeptidase in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and its characteristics after glycosylation

Prolyl aminopeptidases are specific exopeptidases that catalyze the hydrolysis of the N-terminus proline residue of peptides and proteins. In the present study, the prolyl aminopeptidase gene (pap) from Aspergillus oryzae JN-412 was optimized through the codon usage of Pichia pastoris. Both the native and optimized pap genes were inserted into the expression vector pPIC9 K and were successfully expressed in P. pastoris. Additionally, the activity of the intracellular enzyme expressed by the recombinant optimized pap gene reached 61.26 U mL^?1, an activity that is 2.1-fold higher than that of the native gene. The recombinant enzyme was purified by one-step elution through Ni-affinity chromatography. The optimal temperature and pH of the purified PAP were 60 °C and 7.5, respectively. Additionally, the recombinant PAP was recovered at a yield greater than 65 % at an extremely broad range of pH values from 6 to 10 after treatment at 50 °C for 6 h. The molecular weight of the recombinant PAP decreased from 50 kDa to 48 kDa after treatment with a deglycosylation enzyme, indicating that the recombinant PAP was completely glycosylated. The glycosylated PAP exhibited high thermo-stability. Half of the activity remained after incubation at 50 °C for 50 h, whereas the remaining activity of PAP expressed in E. coli was only 10 % after incubation at 50 °C for 1 h. PAP could be activated by the appropriate salt concentration and exhibited salt tolerance against NaCl at a concentration up to 5 mol L^?1.     

Localization of prolyl endopeptidase mRNA in small growing follicles of porcine ovary

Prolyl endopeptidase (EC3.4.21.26) has been considered a unique intracellular enzyme catalyzing internal peptide bond hydrolysis of Pro-X. In this study, the distribution of prolyl endopeptidase activity and its mRNA was investigated in the follicles of porcine ovary. Both follicular fluid and granulosa cell fractions from small follicles showed higher activity than those from large follicles. Molecular cloning and Northern blot analysis suggested that only one species of prolyl endopeptidase gene was expressed in the ovary. In addition, in situ hybridization study revealed that the prolyl endopeptidase mRNA expresssion was more noticeable in the granulosa cell layers of small ovarian follicles than in those of large follicles, suggesting its importance in the early stage of follicular development. Mol. Reprod. Dev. 50:121–127, 1998. © 1998 Wiley-Liss, Inc.     

The purification and characterization of a proline dipeptidase from guinea pig brain

A proline dipeptidase (EC 3.4.13.9) from guinea pig brain was purified to over 90% homogeneity by a combination of ammonium sulfate fractionation, DEAE-cellulose chromatography, calcium phosphate-cellulose chromatography, chromatofocusing, and gel filtration on Sephadex G-200. A purification factor of 2718-fold was obtained with a yield of 7%. The purified enzyme was found to have an apparent molecular weight of 132,000 and to consist of two dissimilar subunits of molecular weights 64,000 and 68,000. The substrate specificity of the enzyme is not that of a strict proline dipeptidase. Although it preferentially hydrolyzes proline dipeptides (Leu-Pro) it also hydrolyzes prolyl dipeptides (Pro-Leu) and dipeptides not containing proline (Leu-Leu). The purified enzyme preparation exhibited weak aminoacylproline aminopeptidase activity against Arg-Pro-Pro but it did not exhibit any post-proline dipeptidyl aminopeptidase, post-proline cleaving endopeptidase, proline iminopeptidase, prolyl carboxypeptidase or carboxypeptidase P activities when tested with a large variety of peptides and arylamides. With all of the proline and prolyl dipeptides examined the enzyme exhibited biphasic kinetics (two distinct slopes on Lineweaver-Burk plots). However, with Leu-Leu as substrate normal Michaelis-Menten kinetics were obeyed.     

Purification and characterization of hemolysin from periodontopathogenic bacterium Eikenella corrodens strain 1073

Eikenella corrodens 1073 was found to show hemolytic activity when grown on sheep blood agar. A high and dose-dependent hemolytic activity was detected in the cell envelope fraction, which was further purified by ion-exchange and gel-filtration chromatography. Consequently, a 65-kDa protein with hemolytic activity was obtained, suggesting that this protein might be a hemolysin. Its N-terminal amino acid sequence was nearly identical to that of X-prolyl aminopeptidase from E. corrodens ATCC 23834. To confirm that X-prolyl aminopeptidase functions as a hemolytic factor, we expressed the hlyA gene, encoding X-prolyl aminopeptidase, in Escherichia coli. After induction with isopropyl β-D-1-thiogalactopyranoside, a protein of about 65 kDa was purified on a Ni column, and its hemolytic activity was confirmed. Meanwhile, a strain with a disrupted hlyA gene, which was constructed by homologous recombination, did not show any hemolytic activity. These results suggested that X-prolyl aminopeptidase might function as a hemolysin in E. corrodens.     

Tyrosinase activity and hemocyanin in the hemolymph of the slipper lobster <Emphasis Type="Italic">Scyllarides latus</Emphasis>

The respiratory protein hemocyanin is present in molluscans and in some species of arthropods, and its dioxygen binding site strongly resembles that of the monophenol-hydroxylating and catechol-quinonising enzyme tyrosinase. Moreover, some hemocyanins show a certain extent of tyrosinase activity, so a common ancestry between the two proteins has been suggested. However, in the case purified hemocyanin of Scyllarides latus any attempts to evoke tyrosinase activity failed. A distinct tyrosinase has been purified to homogeneity from the hemolymph, and kinetically characterised. The purified tyrosinase showed both monophenolase and diphenolase enzyme activity and therefore it could be well defined as a true tyrosinase. This finding suggests that in the case of the studied crustacean the evolutionary functional divergence between dioxygen transport and oxidation of phenolics has already reached its completeness.