Multiple lignin peroxidases of Phanerochaete chrysosporium INA-12 E. Odier and M. Delattre Laboratoire de Microbiologie, Centre de Biotechnologies Agro-Industrielles, Thiverval-Grignon, France
Nine proteins with lignin peroxidase activity were separted from cultures ofPhanerochaete chrysosporium INA-12 in glycerol as carbon source and non-nitrogen limited. Four lignin peroxidase isozymes (4, 5, 8, 9) were purified and characterized. Although differences in kinetic parameters could be shown, antibody reaction showed homology between isozymes. However, thermal stability studied, peptide mapping results, and N-terminal sequence analyses established a higher degree of homology between isozymes 4/5 and 8/9 types. Protein characterization and kinetic data indicate that lignin peroxidase isozymes 4, 5, 8, and 9 differ from described isozymes in strain BKM. The higher specific activity of lignin peroxidase isozymes in cultures with glycerol than in nitrogen-starved cultures accounts for the higher lignin peroxidase activity obtained in these conditions.
Keywords: Ligninperoxidase; Phanerochaete chrysosporium; isozymes
Introduction Lignin-degrading enzymes have potential for applications based on specific degradation of lignin in wood as well as elimination of recalcitrant molecules, r In the white rot fungus Phanerochaete chrysosporium, lignin degradation involves the one-electron oxidation of lignin substructures catalysed by an extracellular HzOzdependent oxidase z,3 later identified as a peroxidase. 4-6 This enzyme, designated lignin peroxidase (ligninase), oxidizes aromatic molecules with subsequent formation of a radical cation. 7 The ability of this new peroxidase to oxidize lignin is related to a higher oxidation potential compared to previously described peroxidases. 8 Multiple lignin peroxidases are secreted in the extracellular medium. 9,1° Walther et al. 11 have presented evidence strongly suggesting the existence of multiple lignin peroxidase genes in P. chrysosporium. Most studies on lignin peroxidase concern P. chrysosporium strain BKM-F-1767. Lignin peroxidases in P. chrysosporium have strong homology based on antibody reactions9,1z; however, differences in kinetic parameters are apparent. 9,1°,~z The main lig-
Abbreviations: TCA, trichloraceticacid; SDS, sodiumdodecylsulfate; ABTS, 2,2'-Azinobis(3-ethylbenzthiazoline-6-sulfonic acid) Address reprint requests to Dr. Odierat the Laboratoirede Microbiologie, Centre de BiotechnologiesAgro-Industrielles,78850Thiverval-Grignon, France Accepted 5 June 1989 © 1990
Butterworth Publishers
nin peroxidase (designated H8) in this strain has been cloned, 13 and the sequence appears to share strong homology around the putative active site with plant peroxidases such as those from horseradish or turnip. 13A lignin peroxidase from strain ME-446 was also cloned, and the sequence showed strong homology with that of H8 in BKM. 14Another type of peroxidase secreted by P. chrysosporium BKM oxidizes Mn(II) to Mn(III).15 This enzyme, designated Mn-peroxidase, does not show lignin peroxidase activity. Multiple forms of Mn-peroxidase have also been separated. 16-17 Lignin peroxidase production is regulated by starvation for nutrient nitrogen or carbon, 18 as is the ligninolytic system in this fungus. 19This regulation has been shown to be at the level of transcription. 13 Distinct lignin peroxidase genes are transcribed in response to limitation by nitrogen or carbon, z° A strain designated INA-12 able to produce lignin peroxidase under non-nitrogen-limiting conditions has been isolated from BKM. zl Lignin peroxidase production in this strain has been enhanced using culture additives consisting essentially of surfactants zz,z3 and lipid sources. 23,z4 Other deregulated mutant strains of P. chrysosporium have been reported from strain ME446. 25,26 Lignin peroxidase isozymes in these mutants differ from those of the parent strain. This study concerns the purification and characterization of lignin peroxidase isozymes in strain INA-12. The homology of isozymes is discussed in connection with possible regulation mechanisms in this species. Enzyme Microb. Technol., 1990, vol. 12, June
447
Papers
Materials and methods: Growth conditions Cultures of Phanerochaete chrysosporium INA-12 (CNCM 398) were grown in non-nitrogen-limited standing cultures with azolectin and oleic acid 24 with the following medium composition (l-i): di-ammonium tartrate 1.84 g (N = 20 mM), KH2PO4 2 g, MgSO4 • 7 H20, CaCI2' 2 H20 0.7 g, FeSO4" 7 HzO 0.07 g, ZnSO4" 7 H20 0.462 g, MnSO4" 7 H20 0.035 g, CuSO4 • 5 H20 0.007 g, glycerol 10 g, 2,2'-dimethylsuccinic acid 1.46 g (pH adjusted to 6.5 with KOH), thiamine 0.0025 g, yeast extract 1 g, oleic acid 0. I g, Tween 80 0.1 g, azolectin 0.75 g, veratryl alcohol 67.28 g. Oleic acid was added as an emulsion with Tween 8023 to which azolectin was added; the lipid mixture was autoclaved before addition to the other medium components. Cultures were grown in 150-ml flasks containing 10 ml medium.
Protein purification Proteins were recovered from the extracellular medium (2 1 total) after filtration of the extracellular fluid through cheesecloth to remove mycelium, centrifugation at 10,000g for 10 min, and concentration by ultrafiltration through a Minitan OM 119 (cutoff 10,000 daltons) (Millipore, Saint Quentin-en-Yvelines, France) until the volume reached 100 ml, after which concentration proceeded using an Amicon (Epernon, France) 8200 PM-10 membrane (cutoff 10,000 daltons). Enzyme concentrates were dialyzed against water and stored at -20°C until purification. Anion exchange chromatography was with a Pharmacia FPLC Mono Q column equilibrated in cacodylate-Na buffer 10 mM pH 5.9. Proteins were eluted by a NaCI gradient from 0 to 1 M in the same buffer. Effluent was recorded at 280 and 405 nm. Peaks were collected and dialyzed against water. Partially purified lignin peroxidases were further purified by Preparative Isoelectric Focusing as described by Leisola9 using Ampholines in the pH range 3.5-5.
Electrophoresis and analytical electrofocusing Proteins were analyzed by SDS-polyacrylamide gel electrophoresis according to Laemmli, z7 modified as follows: Samples dissolved in Tris-HC1 buffer pH 6.8 containing bromophenol blue 0.25% (w/v), SDS 2.5% (w/v), and glycerol 12.5% were applied to the gel at 15 mA per gel (16 cm). The stacking gel was 10% acrylamide, pH 6.8, and the running gel was 10% acrylamide buffer with Tris-HCl pH 8.8 containing 1% SDS. After electrophoresis (7 h), gels were fixed and proteins were stained with Coomassie blue R-250 (LKB application note 250). o~-Lactalbumin (Mr 14,400), trypsin inhibitor (20,100), carbonic anhydrase (30,000), ovalbumin (43,000), bovine serum albumin (67,000), and phosphorylase b (94,000) were used as standards. Analytical isoelectric focusing was performed either according to Bertheau et al. 28 with gels in the range 3.5-5, or using a Pharmacia Phast system apparatus and gels in the range pH 4-6.5. Proteins were visual-
448
Enzyme Microb. Technol., 1990, vol. 12, June
ized in gels using silver staining in the Phast System according to the Pharmacia standard procedure. Peroxidase activity was visualized in gels using benzidine/HzO229 in acetate-Na buffer 0.1 M, pH 3.5. Glycoproteins were visualized in gels using periodic acid staining, z°
Protein characterization
Antibody preparation, Antibodies against lignin peroxidase 8 were raised in rabbits by injecting 100 pg of purified lignin peroxidase in l ml saline buffer mixed with an equal volume of Freund's complete adjuvant (Difco). Booster injection was given with complete adjuvant after 3 weeks and repeated with incomplete adjuvant after 3 weeks. Antibodies specific for lignin peroxidase were detectable in Ouchterlony double diffusion assay 9 weeks after the first injection. Animals were bled and serum was collected. Automated Edman degradation of the native proteins was performed following the original method of Hewick et al. 3° using an Applied Biosystems 470A sequencer and its on-line phenylthiohydantoin amino acid derivative analyzer model 120A with reagents and methods of the manufacturer. Peptide mapping was carried out using cleavage by V8 protease as described by Kirk et al.12 Affinity to concanavalin A-Sepharose was investigated as described by Renganathan et al. 31
Enzyme assays. Lignin peroxidase was assayed using the veratryl alcohol oxidation assay according to Tien and Kirk 32 at pH 3 and 30°C. Peroxidase assay used ABTS. 33 Mn-peroxidase was assayed based on the Poly B-411 decolorization and MnlI oxidation assays. 33 Proteins were measured according to Bradford 34 using bovine serum albumin as a standard. Chemicals and reagents. Veratryl alcohol (Fluka) was purified by vacuum-distillation before use. 1,4-Dimethoxybenzene was from Aldrich. N-Glycanase (peptide:N-glycosidase F) was from Sigma. All sequencing reagents were from applied Biosystems. Results Purification o f lignin peroxidases Cultures of P. chrysosporium INA-12 were harvested after 5 days of incubation when they had reached maximum lignin peroxidase activity (40 nkat ml i). At this time, cultures were neither carbon- nor nitrogen-limited, since the medium contained 7 mM NH~ and 38 mM (3.5 gl i) glycerol. Extracellular proteins were concentrated and fractionated using anion exchange chromatography in a first step. Eight peaks with absorbance at 405 nm (Figure 1) were separated. All showed lignin peroxidase activity according to the veratryl alcohol oxidation assay. Isoelectric focusing established the presence of several proteins in each of the eight peaks. Nine bands could be separated by preparative isoelectric focusing with lignin peroxidase activity.
Lignin peroxidases of INA- 12: E. Odier and M. Delattre 8 7
6
5 4
3 o.s
0.5
1.0
/
o,l
8 ~'
/ 0.4
o9
t o.8
/
A
t
Isozymes 8 and 9 have identical sequences. The first 20 amino acids in these isozymes do not show any homology with the sequence of H8. However, the sequence of the last eight amino acids in isozyme 8 from amino acid 21 is similar to the sequence of isozymes 4 and 5, and of H8 from amino acid 20. Peptide mapping confirmed the strong homology between isozymes 4 and 5, and 8 and 9 (Figure 3).
o.3
o
I
0
~ o,s
m
Nine proteins with lignin peroxidase activity were separted from cultures ofPhanerochaete chrysosporium INA-12 in glycerol as carbon source and non-nitrogen limited. Four lignin peroxidase isozymes (4, 5, 8, 9) were purified and characterized. Although differences in kinetic parameters could be shown, antibody reaction showed homology between isozymes. However, thermal stability studied, peptide mapping results, and N-terminal sequence analyses established a higher degree of homology between isozymes 4/5 and 8/9 types. Protein characterization and kinetic data indicate that lignin peroxidase isozymes 4, 5, 8, and 9 differ from described isozymes in strain BKM. The higher specific activity of lignin peroxidase isozymes in cultures with glycerol than in nitrogen-starved cultures accounts for the higher lignin peroxidase activity obtained in these conditions.
Keywords: Ligninperoxidase; Phanerochaete chrysosporium; isozymes
Introduction Lignin-degrading enzymes have potential for applications based on specific degradation of lignin in wood as well as elimination of recalcitrant molecules, r In the white rot fungus Phanerochaete chrysosporium, lignin degradation involves the one-electron oxidation of lignin substructures catalysed by an extracellular HzOzdependent oxidase z,3 later identified as a peroxidase. 4-6 This enzyme, designated lignin peroxidase (ligninase), oxidizes aromatic molecules with subsequent formation of a radical cation. 7 The ability of this new peroxidase to oxidize lignin is related to a higher oxidation potential compared to previously described peroxidases. 8 Multiple lignin peroxidases are secreted in the extracellular medium. 9,1° Walther et al. 11 have presented evidence strongly suggesting the existence of multiple lignin peroxidase genes in P. chrysosporium. Most studies on lignin peroxidase concern P. chrysosporium strain BKM-F-1767. Lignin peroxidases in P. chrysosporium have strong homology based on antibody reactions9,1z; however, differences in kinetic parameters are apparent. 9,1°,~z The main lig-
Abbreviations: TCA, trichloraceticacid; SDS, sodiumdodecylsulfate; ABTS, 2,2'-Azinobis(3-ethylbenzthiazoline-6-sulfonic acid) Address reprint requests to Dr. Odierat the Laboratoirede Microbiologie, Centre de BiotechnologiesAgro-Industrielles,78850Thiverval-Grignon, France Accepted 5 June 1989 © 1990
Butterworth Publishers
nin peroxidase (designated H8) in this strain has been cloned, 13 and the sequence appears to share strong homology around the putative active site with plant peroxidases such as those from horseradish or turnip. 13A lignin peroxidase from strain ME-446 was also cloned, and the sequence showed strong homology with that of H8 in BKM. 14Another type of peroxidase secreted by P. chrysosporium BKM oxidizes Mn(II) to Mn(III).15 This enzyme, designated Mn-peroxidase, does not show lignin peroxidase activity. Multiple forms of Mn-peroxidase have also been separated. 16-17 Lignin peroxidase production is regulated by starvation for nutrient nitrogen or carbon, 18 as is the ligninolytic system in this fungus. 19This regulation has been shown to be at the level of transcription. 13 Distinct lignin peroxidase genes are transcribed in response to limitation by nitrogen or carbon, z° A strain designated INA-12 able to produce lignin peroxidase under non-nitrogen-limiting conditions has been isolated from BKM. zl Lignin peroxidase production in this strain has been enhanced using culture additives consisting essentially of surfactants zz,z3 and lipid sources. 23,z4 Other deregulated mutant strains of P. chrysosporium have been reported from strain ME446. 25,26 Lignin peroxidase isozymes in these mutants differ from those of the parent strain. This study concerns the purification and characterization of lignin peroxidase isozymes in strain INA-12. The homology of isozymes is discussed in connection with possible regulation mechanisms in this species. Enzyme Microb. Technol., 1990, vol. 12, June
447
Papers
Materials and methods: Growth conditions Cultures of Phanerochaete chrysosporium INA-12 (CNCM 398) were grown in non-nitrogen-limited standing cultures with azolectin and oleic acid 24 with the following medium composition (l-i): di-ammonium tartrate 1.84 g (N = 20 mM), KH2PO4 2 g, MgSO4 • 7 H20, CaCI2' 2 H20 0.7 g, FeSO4" 7 HzO 0.07 g, ZnSO4" 7 H20 0.462 g, MnSO4" 7 H20 0.035 g, CuSO4 • 5 H20 0.007 g, glycerol 10 g, 2,2'-dimethylsuccinic acid 1.46 g (pH adjusted to 6.5 with KOH), thiamine 0.0025 g, yeast extract 1 g, oleic acid 0. I g, Tween 80 0.1 g, azolectin 0.75 g, veratryl alcohol 67.28 g. Oleic acid was added as an emulsion with Tween 8023 to which azolectin was added; the lipid mixture was autoclaved before addition to the other medium components. Cultures were grown in 150-ml flasks containing 10 ml medium.
Protein purification Proteins were recovered from the extracellular medium (2 1 total) after filtration of the extracellular fluid through cheesecloth to remove mycelium, centrifugation at 10,000g for 10 min, and concentration by ultrafiltration through a Minitan OM 119 (cutoff 10,000 daltons) (Millipore, Saint Quentin-en-Yvelines, France) until the volume reached 100 ml, after which concentration proceeded using an Amicon (Epernon, France) 8200 PM-10 membrane (cutoff 10,000 daltons). Enzyme concentrates were dialyzed against water and stored at -20°C until purification. Anion exchange chromatography was with a Pharmacia FPLC Mono Q column equilibrated in cacodylate-Na buffer 10 mM pH 5.9. Proteins were eluted by a NaCI gradient from 0 to 1 M in the same buffer. Effluent was recorded at 280 and 405 nm. Peaks were collected and dialyzed against water. Partially purified lignin peroxidases were further purified by Preparative Isoelectric Focusing as described by Leisola9 using Ampholines in the pH range 3.5-5.
Electrophoresis and analytical electrofocusing Proteins were analyzed by SDS-polyacrylamide gel electrophoresis according to Laemmli, z7 modified as follows: Samples dissolved in Tris-HC1 buffer pH 6.8 containing bromophenol blue 0.25% (w/v), SDS 2.5% (w/v), and glycerol 12.5% were applied to the gel at 15 mA per gel (16 cm). The stacking gel was 10% acrylamide, pH 6.8, and the running gel was 10% acrylamide buffer with Tris-HCl pH 8.8 containing 1% SDS. After electrophoresis (7 h), gels were fixed and proteins were stained with Coomassie blue R-250 (LKB application note 250). o~-Lactalbumin (Mr 14,400), trypsin inhibitor (20,100), carbonic anhydrase (30,000), ovalbumin (43,000), bovine serum albumin (67,000), and phosphorylase b (94,000) were used as standards. Analytical isoelectric focusing was performed either according to Bertheau et al. 28 with gels in the range 3.5-5, or using a Pharmacia Phast system apparatus and gels in the range pH 4-6.5. Proteins were visual-
448
Enzyme Microb. Technol., 1990, vol. 12, June
ized in gels using silver staining in the Phast System according to the Pharmacia standard procedure. Peroxidase activity was visualized in gels using benzidine/HzO229 in acetate-Na buffer 0.1 M, pH 3.5. Glycoproteins were visualized in gels using periodic acid staining, z°
Protein characterization
Antibody preparation, Antibodies against lignin peroxidase 8 were raised in rabbits by injecting 100 pg of purified lignin peroxidase in l ml saline buffer mixed with an equal volume of Freund's complete adjuvant (Difco). Booster injection was given with complete adjuvant after 3 weeks and repeated with incomplete adjuvant after 3 weeks. Antibodies specific for lignin peroxidase were detectable in Ouchterlony double diffusion assay 9 weeks after the first injection. Animals were bled and serum was collected. Automated Edman degradation of the native proteins was performed following the original method of Hewick et al. 3° using an Applied Biosystems 470A sequencer and its on-line phenylthiohydantoin amino acid derivative analyzer model 120A with reagents and methods of the manufacturer. Peptide mapping was carried out using cleavage by V8 protease as described by Kirk et al.12 Affinity to concanavalin A-Sepharose was investigated as described by Renganathan et al. 31
Enzyme assays. Lignin peroxidase was assayed using the veratryl alcohol oxidation assay according to Tien and Kirk 32 at pH 3 and 30°C. Peroxidase assay used ABTS. 33 Mn-peroxidase was assayed based on the Poly B-411 decolorization and MnlI oxidation assays. 33 Proteins were measured according to Bradford 34 using bovine serum albumin as a standard. Chemicals and reagents. Veratryl alcohol (Fluka) was purified by vacuum-distillation before use. 1,4-Dimethoxybenzene was from Aldrich. N-Glycanase (peptide:N-glycosidase F) was from Sigma. All sequencing reagents were from applied Biosystems. Results Purification o f lignin peroxidases Cultures of P. chrysosporium INA-12 were harvested after 5 days of incubation when they had reached maximum lignin peroxidase activity (40 nkat ml i). At this time, cultures were neither carbon- nor nitrogen-limited, since the medium contained 7 mM NH~ and 38 mM (3.5 gl i) glycerol. Extracellular proteins were concentrated and fractionated using anion exchange chromatography in a first step. Eight peaks with absorbance at 405 nm (Figure 1) were separated. All showed lignin peroxidase activity according to the veratryl alcohol oxidation assay. Isoelectric focusing established the presence of several proteins in each of the eight peaks. Nine bands could be separated by preparative isoelectric focusing with lignin peroxidase activity.
Lignin peroxidases of INA- 12: E. Odier and M. Delattre 8 7
6
5 4
3 o.s
0.5
1.0
/
o,l
8 ~'
/ 0.4
o9
t o.8
/
A
t
Isozymes 8 and 9 have identical sequences. The first 20 amino acids in these isozymes do not show any homology with the sequence of H8. However, the sequence of the last eight amino acids in isozyme 8 from amino acid 21 is similar to the sequence of isozymes 4 and 5, and of H8 from amino acid 20. Peptide mapping confirmed the strong homology between isozymes 4 and 5, and 8 and 9 (Figure 3).
o.3
o
I
0
~ o,s
m
