A Novel White Laccase from Pleurotus ostreatus

Abstract

Two laccase isoenzymes (POXA1 and POXA2) produced by Pleurotus ostreatus were purified and fully characterized. POXA1 and POXA2 are monomeric glycoproteins with 3 and 9% carbohydrate content, molecular masses of about 61 and 67 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, of about 54 and 59 kDa by gel filtration in native conditions, and of 61 kDa by matrix-assisted laser desorption ionization mass spectrometry (only for POXA1) and pI values of 6.7 and 4.0, respectively. The N terminus and three tryptic peptides of POXA1 have been sequenced, revealing clear homology with laccases from other microorganisms, whereas POXA2 showed a blocked N terminus. The stability of POXA2 as a function of temperature was particularly low, whereas POXA1 showed remarkable high stability with respect to both pH and temperature.Both enzymes oxidize syringaldazine and ABTS (2, 2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) together with a variety of different substituted phenols and aromatic amines with the concomitant reduction of oxygen, but POXA1 is unable to oxidize guaiacol. Both enzymes were strongly inhibited by sodium azide and thioglycolic acid but not by EDTA.UV/visible absorption spectra, atomic adsorption, and polarographic data indicated the presence of 4 copper atoms/mol of POXA2 but only one copper, two zinc, and one iron atoms were found/mol of POXA1.The neutral pI and the anomalous metal content of POXA1 laccase render this enzyme unique in its structural characteristics. The lack of typical absorbance at 600 nm allows its classification as a “white” laccase. Two laccase isoenzymes (POXA1 and POXA2) produced by Pleurotus ostreatus were purified and fully characterized. POXA1 and POXA2 are monomeric glycoproteins with 3 and 9% carbohydrate content, molecular masses of about 61 and 67 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, of about 54 and 59 kDa by gel filtration in native conditions, and of 61 kDa by matrix-assisted laser desorption ionization mass spectrometry (only for POXA1) and pI values of 6.7 and 4.0, respectively. The N terminus and three tryptic peptides of POXA1 have been sequenced, revealing clear homology with laccases from other microorganisms, whereas POXA2 showed a blocked N terminus. The stability of POXA2 as a function of temperature was particularly low, whereas POXA1 showed remarkable high stability with respect to both pH and temperature. Both enzymes oxidize syringaldazine and ABTS (2, 2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) together with a variety of different substituted phenols and aromatic amines with the concomitant reduction of oxygen, but POXA1 is unable to oxidize guaiacol. Both enzymes were strongly inhibited by sodium azide and thioglycolic acid but not by EDTA. UV/visible absorption spectra, atomic adsorption, and polarographic data indicated the presence of 4 copper atoms/mol of POXA2 but only one copper, two zinc, and one iron atoms were found/mol of POXA1. The neutral pI and the anomalous metal content of POXA1 laccase render this enzyme unique in its structural characteristics. The lack of typical absorbance at 600 nm allows its classification as a “white” laccase. White rot Basidiomycetes are microorganisms able to efficiently degrade lignin. However the different degradation degree of lignin with respect to other wood components depends very much on the environmental conditions and the fungal species involved. It is now clear that there is not a unique mechanism to achieve the process of lignin degradation and that the enzymatic machinery of the various microorganisms are different (1Hatakka A. FEMS Microbiol. Rev. 1994; 13: 125-135Crossref Scopus (880) Google Scholar). Pleurotus ostreatus belongs to a subclass of lignin-degrading microorganisms that produce laccases, manganese peroxidases, and veratryl alcohol oxidases but no lignin peroxidases. The oxidative enzymes (laccase, manganese peroxidase) do catalyze the formation of radical intermediates from high molecular weight lignins, but the intermediates produced can recondense, shifting back the reaction course (2Hammel K.E. Jensen Jr., K.A. Mozuch M.D. Landucci L.L. Tien M. Pease E.A. J. Biol. Chem. 1993; 268: 12274-12281Abstract Full Text PDF PubMed Google Scholar). It has been shown that prevention of the repolymerization is achieved by the reduction of the formed radicals; this reducing process is carried out by some flavin adenine dinucleotide-dependent oxidases such as veratryl alcohol oxidase (3Marzullo L. Cannio R. Giardina P. Santini M.T. Sannia G. J. Biol. Chem. 1995; 270: 3823-3827Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Laccases and manganese peroxidases are able to oxidize only phenolic residues of lignin, whereas lignin peroxidases have been shown to be effective in the oxidation of the nonphenolic residues of the polymer (4Kirk T.K. Farrell R.L. Annu. Rev. Microbiol. 1987; 41: 465-505Crossref PubMed Scopus (2049) Google Scholar). However, in the presence of mediators, the substrate range of laccases can be extended (5Bourbonnais R. Paice M.G. FEBS Lett. 1990; 267: 99-102Crossref PubMed Scopus (1204) Google Scholar). Laccases have been isolated from various fungi (6Yaropolov A.I. Skorobogatko O.V. Vartanov S.S. Varfolomeyev S.D. Appl. Biochem. Biotechnol. 1994; 49: 257-280Crossref Scopus (609) Google Scholar). They belong to the class of the blue oxidases containing 4 copper atoms/molecule distributed in three different copper binding sites (7Reinhammar B. Malstrom B.G. Spiro T.G. Copper Proteins (Metal Ions in Biology). John Wiley & Sons, Inc., New York1981: 109-149Google Scholar, 8Solomon E.I. Sundaram U.M. Machonkin T.E. Chem. Rev. 1997; 96: 2563-2605Crossref Scopus (3161) Google Scholar). The type 1 (or blue copper) site is responsible for the intense blue color of the enzymes, presumably due to a ligand-to-metal charge transfer absorption involving cysteine sulfur and Cu(II). The type 2 copper exhibits lower visible absorbance, and the type 3 site incorporates two copper centers and is responsible for a shoulder near 330 nm in the absorbance spectrum of native laccase. All these copper ions are apparently involved in the catalytic mechanism. The laccase reduces oxygen to water and simultaneously performs a one electron oxidation of many aromatic substrates (polyphenols, methoxy-substituted monophenols, aromatic amines, etc.). The enzyme is present in multiple isoforms in almost all fungal species, including P. ostreatus (9Giardina P. Cannio R. Martirani L. Marzullo L. Palmieri G. Sannia G. Appl. Environ. Microbiol. 1995; 61: 2408-2413Crossref PubMed Google Scholar). Studies of the genes coding for these enzymes in P. ostreatus have led to the identification of two different genes and two corresponding cDNAs. One of these genes codes for the isoenzyme produced most abundantly under all the growth conditions examined (10Giardina P. Aurilia V. Cannio R. Marzullo L. Amoresano A. Siciliano R. Pucci P. Sannia G. Eur. J. Biochem. 1996; 235: 508-515Crossref PubMed Scopus (91) Google Scholar). In the present paper, we report the purification and the physico-chemical and catalytic properties of two different laccase isoenzymes isolated from P. ostreatus. One of these proteins shows peculiar differences with regard to copper content. This is the first laccase having 2 zinc, 1 iron, and only 1 copper atom/molecule. White rot fungus P. ostreatus (strain Florida) was maintained through periodic transfer at 4 °C on potato dextrose agar plates (Difco) in the presence of 0.5% yeast extract (Difco). Incubations were carried out at 25 °C in the dark by preinoculating 300 ml of potato dextrose broth (24 g/l) containing 0.5% yeast extract in 500 ml shaken flasks with the P. ostreatus mycelia. 50 ml of a 5-day-old culture were transferred in 1 liter flasks containing 450 ml of broth. The cultures were incubated in the dark at 25 °C on a rotary shaker (100 rev/min). At different incubation times, the medium was collected and filtered through gauze. Proteins were precipitated from the filtered medium by the addition of (NH4)2SO4 up to 80% saturation at 4 °C and centrifuged at 10,000 × g for 30 min. The precipitate was resuspended in 50 mm sodium phosphate buffer, pH 6.0, and extensively dialyzed against the same buffer. The sample was again centrifuged, and the supernatant, concentrated on an Amicon PM-10 membrane, was loaded on DEAE-Sepharose Fast Flow (Pharmacia Biotech Inc.) column (1,5 × 40 cm) equilibrated with the phosphate buffer. The column was washed at a flow rate of 30 ml/h with 400 ml of buffer, and a 0–0.5 m NaCl linear gradient (500 ml) was applied. Fractions containing phenol oxidase activity were pooled and concentrated on an Amicon PM-10 membrane. The phenol oxidase POXA1 was then equilibrated in 0.1 mcitrate buffer, pH 5 (buffer A), with a Centricon 30 microconcentrator and loaded onto an ion exchange Mono S HR 5/5 column in fast protein liquid chromatography (Pharmacia) equilibrated with the same buffer. The enzyme was eluted with a linear gradient (buffer B: 0.1m citrate, pH 5, 0.3 m NaCl; gradient:t = 0, %B = 0; t = 10 min, %B = 0; t = 30 min, %B = 80;t = 35 min, %B = 80). The active fractions were pooled and desalted. The phenol oxidase POXA2 was equilibrated in 0.02 m sodium phosphate buffer, pH 7 (buffer A), and loaded onto a cationic exchange Mono Q HR 5/5 column in a fast protein liquid chromatography system equilibrated with the same buffer. The enzyme was eluted with a linear gradient (buffer B, 0.02 m sodium phosphate buffer, pH 7, 0.5 m NaCl; gradient: t = 0, %B = 0;t = 10 min, %B = 0; t = 60 min, %B = 60; t = 65 min, %B = 80). The active fractions were pooled and desalted. Phenol oxidase activity was assayed at 25 °C using 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) 1The abbreviations used are: ABTS, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); DMP, 2,6-dimethoxyphenol; PAGE, polyacrylamide gel electrophoresis., guaiacol, 2,6-dimethoxyphenol (DMP), and syringaldazine as substrates as follows. (a) The assay mixture contained 2 mm ABTS and 0.1 m sodium citrate buffer, pH 3.0. Oxidation of ABTS was followed by absorbance increase at 420 nm (ε = 36,000m−1 cm−1). Enzyme activity was expressed in international units (IU). (b) The assay mixture contained 10 mm guaiacol, and the McIlvaine's citrate-phosphate buffer adjusted to pH 6.0. Oxidation was followed by the absorbance increase at 460 nm. One unit of activity is the amount of enzyme producing a 1.0 A increase/min. (c) The assay mixture contained 1 mm DMP and the McIlvaine's buffer adjusted to pH 5 for POXA1 and POXC isoenzyme and pH 6.5 for POXA2 isoenzyme. Oxidation of DMP was followed by an absorbance increase at 477 nm (ε = 14,800 m−1cm−1). Enzyme activity was expressed in IU. (d) The assay mixture contained 0.5 mm syringaldazine (dissolved in ethanol) and 50 mm phosphate buffer, pH 6. Oxidation of syringaldazine was followed by an absorbance increase at 525 nm (ε = 65,000 m−1 cm−1). Enzyme activity was expressed in IU. Deoxygenase activity was assayed at 30 °C using the protocatechuic acid as substrate as follow. The reaction mixture contained 0.6 mm protocatechuic acid and 50 mm Tris/HCl buffer, pH 8.0. After 10 min of incubation at 30 °C, the reaction was stopped by adding 0.5 ml of 30% perchloric acid. In the control, perchloric acid was added at zero time. The deoxygenation of protocatechuic acid to β-carboxy-cis,cis-muconic acid was followed by an absorbance decrease at 290 nm (11Wojtas-Wasilewska M. Trojanowski J. Luterek J. Acta Biochim. Pol. 1983; 30: 291-302PubMed Google Scholar). Tyrosinase activity was assayed at 25 °C using 3,4-dihydroxy-dl-phenylalanine as substrate. The assay mixture contained 0.2 mm4-dihydroxy-dl-phenylalanine and 0.1 sodium phosphate, pH 6. The oxidation of substrate was followed by an absorbance increase at 475 nm (ε = 3,600 m−1 cm−1) (12Fling M. Horowitz N.H. Heinemann F.S. J. Biol. Chem. 1963; 238: 2045-2053Abstract Full Text PDF PubMed Google Scholar). A peroxidase-coupled assay was using as the substrate. The reaction mixture contained 0.1 citrate buffer, pH 0.3 of mm 2 mm ABTS in a of 1 The oxidation of was followed by an absorbance increase at 460 nm Biochem. PubMed Scopus Google Scholar). Phenol oxidase activity as a function of pH was using a McIlvaine's citrate-phosphate buffer adjusted to different pH in the range The same buffer was used to the pH stability of the three The of various were by using ABTS as a substrate and the isoenzymes for 5 min at temperature the addition of substrate. The activity of phenol oxidases different substrates was assayed in 50 mm sodium phosphate buffer, pH 6.0, the absorbance decrease at the indicated and at 0.1 mm substrate acid acid acid acid acid J. L. R. Biotechnol. Appl. Biochem. 1995; Google Scholar). The absorption of native isoenzymes were and nm at temperature in 50 mm sodium phosphate buffer, pH 6.0, using a was using the protein assay with as a The molecular masses of native phenol oxidases were with a system (Pharmacia) by using a gel filtration column The column was eluted with 50 mm sodium phosphate buffer, pH containing mm and the was at and nm rate The of the column was with and as laser desorption ionization mass were with a linear and 1 of as the were loaded onto the sample and the were using and for of POXA1 POXA2 laccases was as by J. L. R. Biotechnol. Appl. Biochem. 1995; Google 10 of protein in 30 of water were with 2 of and with 25 of a containing 50 mm pH and mm EDTA. The mixture was for 5 min, then 1 unit of was and the reaction mixture was incubated for at 30 and glycoproteins with the were onto an and binding to with the used were the for for acid for acid for and for The protein to the was by a The was to the gel in was carried out as by PubMed Scopus Google Scholar). molecular mass the gel was with and Proteins were by in the pH range was on gel with a system (Pharmacia) the Proteins were using the Phenol oxidase activity was the gel at 25 °C in 0.1 mcitrate buffer, pH containing 2 mm ABTS using 10 mm as The acid of purified phenol oxidases was by degradation with an liquid protein with a high liquid chromatography for the acid Proteins by were on a membrane, and the were was by of the proteins in pH with at °C was carried out on a column × eluted with linear gradient of in acid at a flow rate of 0.2 copper, and iron were in and on different enzyme by atomic absorption spectrometry using a with and of the protein sample (100 were the addition of 2 ml of an mixture of The was resuspended with 1 ml of and then to 25 ml with ml were for polarographic using a with an have been by the with a of 60 at A course of phenol oxidase activity in P. ostreatus culture broth is shown in 1 The activity a at about and of from the at different growth indicated that the activity was with the of two isoenzymes POXA1 and the of POXA1 were to to the of the phenol oxidase activity in the culture broth. In no differences in the amount of the POXC isoenzyme were at different growth P. of was by sulfate followed by exchange shown in different phenol oxidase fractions and POXC were A of phenol oxidase activity and a containing a phenol oxidase isoenzyme were with the buffer, whereas the other three and were eluted with a gradient at and m respectively. Fractions corresponding to the POXA1 isoenzyme were collected and purified by cationic exchange chromatography in a 0.1m citrate buffer at pH The enzyme was eluted as a with a gradient at about 0.1 The POXA2 isoenzyme was on a Mono Q column equilibrated in mm sodium phosphate buffer, pH and eluted in the fractions corresponding to m The two purified POXA1 and to be by and gel filtration The two phenol and were not whereas the POXC isoenzyme was to to the fully enzyme (10Giardina P. Aurilia V. Cannio R. Marzullo L. Amoresano A. Siciliano R. Pucci P. Sannia G. Eur. J. Biochem. 1996; 235: 508-515Crossref PubMed Scopus (91) Google Scholar, G. Giardina P. Marzullo L. B. G. Cannio R. Sannia G. Appl. Microbiol. Biotechnol. 1993; PubMed Scopus Google Scholar). A of the purification is shown in purification was achieved for POXA1 isoenzyme with a of whereas a lower for POXA2 isoenzyme was due to the of activity the purification The of POXA1 and POXA2 are × and of laccases from Pleurotus ostreatus × × × × × × × × × × × × × × × × in a The molecular masses by were 61 kDa and 67 kDa for POXA1 and respectively. A of the POXA1 molecular mass was by mass a at was filtration chromatography of the native enzymes the of molecular mass of 54 and 59 kDa for POXA1 and respectively. the monomeric of these The of POXA1 and POXA2 are 6.7 and 4.0, respectively. POXA1 and POXA2 were with and by Proteins in the gel to corresponding to molecular masses of 59 and 61 kDa a 3 and 9% carbohydrate content, respectively. POXA1 and POXA2 were for the presence of by binding Both proteins were by G. to the presence of high with M. and the of both and containing the at In the N terminus and three tryptic of POXA1 isoenzyme are shown and with the of other laccases (9Giardina P. Cannio R. Martirani L. Marzullo L. Palmieri G. Sannia G. Appl. Environ. Microbiol. 1995; 61: 2408-2413Crossref PubMed Google Scholar, P. Aurilia V. Cannio R. Marzullo L. Amoresano A. Siciliano R. Pucci P. Sannia G. Eur. J. Biochem. 1996; 235: 508-515Crossref PubMed Scopus (91) Google Scholar, A. J. M. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar, R. P. Appl. Environ. Microbiol. 1993; PubMed Google Scholar, L. Biochim. 1995; PubMed Scopus Google Scholar, M. J. Microbiol. PubMed Scopus Google Scholar, M. J. Microbiol. 1993; PubMed Scopus Google Scholar). the N terminus of POXA2 from in on were a blocked N terminus for this isoenzyme. the of the purified enzymes were and with that of the POXC a different was In the spectrum of POXC showed a at nm and a shoulder at 330 nm these are typical of the oxidase enzymes corresponding to type 1 blue copper and to type 3 copper The to A is is to of laccases from other J. L. R. Biotechnol. Appl. Biochem. 1995; Google Scholar, T.K. Appl. Environ. Microbiol. 1995; 61: PubMed Google Scholar, Appl. Environ. Microbiol. 1996; PubMed Google Scholar). In in the POXA1 absorption the was as by the concentrated of the whereas a at about 400 nm was The spectrum of the POXA2 isoenzyme showed a shoulder at 400 nm and a intense absorption at nm with of The copper content was by atomic The POXA2 and POXC isoenzymes showed values of 0.1 and 0.5 whereas only 0.2 of of protein was for POXA1. different of POXA1 isoenzyme were examined for metal content iron, by atomic the presence of two other iron and zinc, was a in a 0.2 and 0.2 values a of for POXA1 isoenzyme. of POXA1 and POXC the presence of copper and be by this for the POXA1 isoenzyme and only the presence of copper for the POXC isoenzyme. using POXC that the three isoenzymes are not The of POXA1 and POXA2 at different pH values was examined different incubation and with that of POXA1 was the most enzyme at all pH values 4.0, 6.0, the stability of this enzyme is almost by pH = at pH in with the other two to be very to pH decrease 2 for POXA2 and 30 min for POXC at pH and lower of all isoenzymes were at pH all of showed of the activity incubation at 60 °C indicated a t of min for 30 min for and 10 min for POXA2 the activity of these enzymes was as a function of the POXA1 showed activity in the range °C, whereas POXC showed activity in a range and at a lower temperature The catalytic of POXA1 and POXA2 with respect to substrates guaiacol, DMP, and were and with of POXC The of POXA2 have not been of the high of this not a of the of the active of the of Pleurotus ostreatus laccase min × × × × × × × in a activity of POXA1 against was at of the pH shown in POXA2 2,6-dimethoxyphenol at pH in this respect from the other two the oxidative reaction by the POXA1 a of different substrates were under The enzyme and and are substrates of all In POXA1 efficiently phenols and with phenols and POXA1 not and It has been that both POXA1 and POXA2 the reaction that catalyze and that no formation be The of laccase on the activity of all the isoenzymes were examined using ABTS as substrate at pH 3.0. All enzymes were inhibited by 0.02 mm sodium azide and by thioglycolic acid at In the presence of no was up to 50 mm for all the three 5 of POXA1 and whereas was to a of 50 mm to the same for the enzymes were acid was in was at and 40 mm for and POXC respectively. This and (9Giardina P. Cannio R. Martirani L. Marzullo L. Palmieri G. Sannia G. Appl. Environ. Microbiol. 1995; 61: 2408-2413Crossref PubMed Google Scholar, P. Aurilia V. Cannio R. Marzullo L. Amoresano A. Siciliano R. Pucci P. Sannia G. Eur. J. Biochem. 1996; 235: 508-515Crossref PubMed Scopus (91) Google Scholar, G. Giardina P. Marzullo L. B. G. Cannio R. Sannia G. Appl. Microbiol. Biotechnol. 1993; PubMed Scopus Google that the of multiple laccase isoforms in the rot fungus P. ostreatus. In we in P. ostreatus two genes and the corresponding coding for two laccase the of one of these is the isoenzyme most abundantly produced in all growth conditions examined (10Giardina P. Aurilia V. Cannio R. Marzullo L. Amoresano A. Siciliano R. Pucci P. Sannia G. Eur. J. Biochem. 1996; 235: 508-515Crossref PubMed Scopus (91) Google Scholar, G. Giardina P. Marzullo L. B. G. Cannio R. Sannia G. Appl. Microbiol. Biotechnol. 1993; PubMed Scopus Google whereas the protein by the other has not been (9Giardina P. Cannio R. Martirani L. Marzullo L. Palmieri G. Sannia G. Appl. Environ. Microbiol. 1995; 61: 2408-2413Crossref PubMed Google Scholar). the of the laccase isoenzymes produced by P. we isolated and two other phenol oxidases (POXA1 and POXA2) from this The course of POXA1 showed differences with respect to that of POXC laccase. The POXA1 activity was the fungal growth with respect to that of a fast decrease of the POXA1 activity was due to the presence in the culture medium of a molecular weight not This a different for the two The other was produced in lower at all growth POXA2 exhibits to of laccases from other in molecular metal content, and the range for other laccases 1994; Scopus Google Scholar). However, the stability of POXA2 as a function of temperature was particularly This to a of the activity the purification a very of the POXA1 isoenzyme shows a remarkable high stability with respect to both pH and temperature and with that of POXC and of other peculiar of POXA1 is its neutral pI to the of all the laccases purified and showed a pI in a pH range from 3 to However, the pI is not the most of this in concentrated of POXA1 lack the typical blue color that all the blue This was by the of the UV/visible spectrum of the the of the type copper the metal content of the protein was both by atomic absorption and by a was The protein was to only 1 copper of the and 2 atoms and 1 iron were protein the that the enzyme belongs to the laccase is by the high degree of of the with the corresponding of laccases, and the of as oxidative substrate and the lack of formation of as a in the the almost of substrate by this enzyme with that of other The data clear that the POXA1 laccase ostreatus belongs to the laccase but structural that render unique and its classification as a laccase. is to the mechanism of the oxidative reaction by this enzyme and the of the metal ions present in this The to for polarographic