INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

White rot basidiomycetes have received extensive attention because of their lignin-degrading activity. The biochemistry of lignin degradation is a complex process involving a series of enzymatic and nonenzymatic reactions. Extracellular enzymes which catalyze oxidative reactions are lignin peroxidases, laccases, manganese peroxidases, and hydrogen peroxide-producing enzymes (3, 13). Even though their catalyzed reactions have been studied in detail, their in vivo coordination and, possibly, synergistic action are not clearly understood.

Pleurotus ostreatus is a white rot basidiomycete which belongs to the subclass of ligninolytic microorganisms that produce laccases, manganese peroxidases, and veratryl alcohol oxidases but no lignin peroxidase. Among these enzymes, laccases have been the most widely studied and characterized (11, 12, 17). In a recent study (16), it has been demonstrated that a laccase isoenzyme (POXA1b; phenol oxidase A1b) is specifically degraded in the early phase of fungal growth by proteases present in P. ostreatus culture broth; hence, the disappearance of POXA1b seems to be correlated with the appearance of extracellular protease activity. A similar relationship was observed for lignin peroxidases in Phanerochaete chrysosporium, and, in this case, the extracellular proteases caused an almost complete disappearance of lignin peroxidase activity due to degradation of all lignin peroxidase isoenzymes (8). Furthermore, a recent report (21) suggests that both intracellular and extracellular proteases are involved in the regulation of ligninolytic activities in cultures of Trametes versicolor under nutrient limitation. In contrast, it has been reported (2) that proteases are not responsible for the decrease in peroxidase activity in Pleurotus pulmonarius cultures. Moreover, a purified protease from solid substrate cultures of Phanerochaete chrysosporium did not affect lignin peroxidase (5). Hence, it is not clear if there is a relationship between ligninolytic activity and protease secretion in white rot fungi.

Despite the fact that the production of extracellular proteolytic enzymes is a common feature among fungi, relatively few proteases secreted from lignin-degrading fungi have been characterized on a molecular level (5, 9, 15). This paper reports the purification and characterization of a novel protease (named PoSl) present in liquid culture of P. ostreatus which appears to belong to the serine protease family. Its structural and kinetic properties are significantly different from those of other proteases purified from P. ostreatus fruiting bodies (7). The purified enzyme is involved in POXA1b degradation and in activation of another recently characterized laccase isoenzyme (unpublished data). On the basis of these results, we hypothesize that extracellular proteases could play a regulatory role in laccase activity in P. ostreatus.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Organism and culture conditions. The white rot fungus P. ostreatus (Jacq.:Fr.) Kummer (type:Florida) was maintained through periodic transfer at 4°C on potato dextrose agar plates (Difco) in the presence of 0.5% yeast extract (Difco).

Enzyme purification. Proteins were precipitated from 3 liters of filtered medium supplemented with CuSO₄ plus FeCl₃ 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 7.0, 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 a DEAE-Sepharose Fast Flow (Pharmacia Biotech Inc.) column (1.5 by 40 cm) equilibrated with the phosphate buffer. The column was washed at a flow rate of 30 ml/h with 150 ml of buffer, and a 0 to 0.5 M NaCl linear gradient (200 ml) was applied. Fractions containing protease activity were pooled and concentrated on an Amicon PM-10 membrane.

Enzyme assays. Protease activity was assayed using N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (SucAAPFpNA), N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide (SucAAPLpNA), azo dye-impregnated collagen (Azocoll) , N-p-tosyl-L-arginine methyl ester (TAME), and N-benzoyl-L-tyrosine ethyl ester (BTEE), all from Sigma, as substrates as follows.

(i) SucAAPFpNA as substrate. The SucAAPFpNA assay mixture contained 5 mM SucAAPFpNA, 10 mM CaCl₂, and 50 mM Tris-HCl buffer, pH 8.0, in a final volume of 1 ml. Hydrolysis of the substrate was monitored by noting the increase in absorbance at 405 nm (₄₀₅ = 8,800 M¹ cm¹).

(ii) SucAAPLpNA as substrate. The SucAAPLpNA assay mixture contained 5 mM SucAAPLpNA, 10 mM CaCl₂, and 50 mM Tris-HCl buffer, pH 7.0, in a final volume of 1 ml. Hydrolysis of the substrate was followed by an increase in absorbance at 405 nm (₄₀₅ = 8,800 M¹ cm¹).

(iii) Azocoll as substrate. The protease activity assay using Azocoll as the substrate was performed as described by Datta (5). One unit of activity was defined as the amount of enzyme that catalyzed the release of enough azo dye to give a change in absorbance at 520 nm of 0.001 in 30 min.

(iv) TAME as substrate. The TAME assay mixture contained 10 mM TAME, 10 mM CaCl₂, and 50 mM Tris-HCl buffer, pH 8.0, in a final volume of 1 ml. Hydrolysis of TAME was monitored at room temperature by noting the increase in absorbance at 247 nm (₂₄₇ = 540 M¹ cm¹).

(v) BTEE as substrate. The BTEE assay mixture contained 0.5 mM BTEE, 10 mM CaCl₂, and 50 mM Tris-HCl buffer, pH 8.0, in a final volume of 1 ml. Hydrolysis of BTEE was monitored at room temperature by noting the increase in absorbance at 256 nm (₂₅₆ = 964 M¹ cm¹).

Protein determination. Protein concentration was determined using the Bio-Rad protein assay with bovine serum albumin as the standard.

Nondenaturing PAGE. Polyacrylamide gel electrophoresis (PAGE) was performed at an alkaline pH under nondenaturing conditions. The separating and stacking gels contained 9 and 4% concentrations of acrylamide, respectively. The buffer solutions were 50 mM Tris-HCl (pH 9.5) for the separating gel and 18 mM Tris-HCl (pH 7.5) for the stacking gel; the electrode reservoir solution was 25 mM Tris-190 mM glycine (pH 8.4). Gels were stained for laccase activity using ABTS [2,2'-azinobis-(3-ethylbenzthiazolinesulfonic acid)] as the substrate as previously described (16).

Determination of molecular mass. The molecular mass of native protease was determined using a Superdex 200 PC 3.2/30 gel filtration column (Pharmacia). The column was eluted with 50 mM sodium phosphate buffer, pH 7.0, containing 150 mM NaCl (flow rate, 1 ml/min). The calibration of the column was performed with alcohol dehydrogenase (150 kDa), hemoglobin (64 kDa), ovalbumin (45 kDa), and carbonic anhydrase (29 kDa) as standards.

Electrophoresis and isoelectrofocusing. Polyacrylamide (9%) gel slab electrophoresis in 0.1% sodium dodecyl sulfate (SDS) was carried out as described by Laemmli (14). For molecular mass determinations, the gel was calibrated with rabbit muscle myosin (205 kDa), -galactosidase (116 kDa), rabbit muscle phosphorylase b (97.4 kDa), bovine serum albumin (66 kDa), egg albumin (45 kDa), and carbonic anhydrase (29 kDa). Proteins were visualized by Coomassie blue staining.

Sequence analysis. Automated N-terminal degradation of the electroblotted protein was performed with a Perkin-Elmer Applied Biosystems 477A pulsed-liquid protein sequencer equipped with a model 120A phenylthiohydantoin analyzer for the online identification and quantification of phenylthiohydantoin amino acids.

Lectin assay. PoSl and control standard glycoproteins (supplied with the Boehringer Mannheim glycan differentiation kit; 1 µg) were directly spotted onto an Immobilon membrane and detected immunologically after binding to lectins conjugated with digoxigenin by following the manufacturer's instructions (Boehringer Mannheim). The lectins used were the following: Galanthus nivalis agglutinin, specific for terminal mannose; Sambucus nigra agglutinin, specific for sialic acid (2-6)-galactose; Maackia amurenais agglutinin, specific for sialic acid (2-3)-galactose; peanut agglutinin, specific for galactose (1-3)-N-acetylglucosamine; and Datura stramonium agglutinin, specific for galactose (1-4)-N-acetylglucosamine. The protein linked to the lectin was detected by a colorimetric reaction. The immunological detection was performed according to the manufacturer's instructions. This experiment was performed in duplicate on two different enzymatic preparations.

MALDI-MS analysis. Matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) analyses were carried out with a Voyager DE MALDI-time of flight MS (PerSeptive Biosystem, Boston, Mass.).

Enzymatic hydrolysis. Protease samples (1 nmol) were reduced by incubation with 200 nmol of dithiothreitol at 37°C for 2 h under a nitrogen atmosphere in 0.25 M Tris-HCl (pH 8.5)-1.25 mM EDTA containing 6 M guanidinium chloride. Free SH groups were alkylated by using an excess of iodoacetamide at room temperature in the dark under a nitrogen atmosphere. Protein samples were freed from salt and an excess of reagent by loading the reaction mixture onto a PD-10 prepacked column (Pharmacia) equilibrated and eluted in 0.4% ammonium bicarbonate, pH 8.5.

Oligosaccharide derivatization. Oligosaccharides were permethylated as described by Dell (6) and directly analyzed by MALDI-MS by mixing permethylated oligosaccharides with 2,5-dihydroxybenzoic acid on a sample plate and drying the plate in vacuo. Mass range was calibrated with bovine insulin (average molecular mass, 5,734.6 Da).

Specificity assay with oxidized B chain of insulin. Oxidized B chain from bovine insulin (Sigma) was hydrolyzed with PoSl, trypsin (bovine pancreas), subtilisin bacterial protease nagarse, chymotrypsin (bovine pancreas), and elastase (pig pancreas), all from Sigma. Incubations were performed in 0.4% ammonium bicarbonate, pH 8.5, at 37°C for 5 min, using an enzyme/substrate ratio of 1:1,000 (wt/wt). Proteolytic mixtures were analyzed at each time by MALDI-MS analysis.

Autocatalytic assay. PoSl autocatalytic activity was checked using 2 µM solutions of PoSl incubated in 0.4% ammonium bicarbonate, pH 8.5, at 37°C. Aliquots were withdrawn at different incubation times (0, 1 h, 3 h, and 18 h), and catalytic activity was stopped by adding 20% trifluoroacetic acid (TFA).

Incubation of PoSl with laccase isoenzymes. A sample of P. ostreatus culture broth supplemented with copper and iron (pH ~6) was collected after 2 days of growth, filtered, and incubated overnight at 25°C with PMSF (1 mM final concentration). After this treatment, no protease activity, using SucAAPFpNA as the substrate, was detected. Fifty microliters of this sample (0.15 U of total laccase activity) was incubated with 0.01 nmol of PoSl in the presence of 10 mM CaCl₂. Aliquots of this sample (12 µl) after 1, 3, and 18 h were analyzed by nondenaturing PAGE.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Protease assay. In order to identify the best substrate for detection of protease activity in P. ostreatus culture broth, four different protease substrates were tested. Activities of 10 and 0.03 U/ml were determined in the basal medium after 3 days of growth using Azocoll and SucAAPFpNA as substrates, respectively (the activity towards Azocoll is expressed in arbitrary units), while no activity was detected with TAME or BTEE as the substrate. Because the SucAAPFpNA assay allows the expression of protease activity in international units, SucAAPFpNA was used as the substrate to establish the catalytic parameters.

Protease purification. Culture conditions which induce protease production were analyzed; two- and threefold-higher protease activities were obtained in the presence of 0.15 mM CuSO₄ and 0.15 mM CuSO₄ plus 0.1 mM FeCl₃, respectively, after 3 days of incubation, than in the basal medium. No increase of protease activity was observed when ZnSO₄ or FeCl₃ was used. Culture broth supplemented with CuSO₄ plus FeCl₃ was fractionated after 3 days of growth by ammonium sulfate precipitation followed by anion-exchange chromatography at pH 7.0. Three different protease fractions were separated. A major peak (PoSl) was eluted with a saline gradient at approximately 0.4 M NaCl. Fractions corresponding to PoSl were collected and further purified by gel filtration chromatography (Superdex 75) and anion-exchange chromatography on a Mono Q column at pH 7.0. The purified protein appeared to be homogeneous when analyzed by SDS-PAGE, isoelectric focusing, and gel filtration chromatography. A summary of the purification procedure is shown in Table 1; about 70-fold purification was achieved, with a final yield of 13%.

Physical and chemical properties. The molecular mass of PoSl is 75 kDa under denaturing conditions (SDS-PAGE) and 74 kDa under native conditions (gel filtration chromatography), thus indicating the monomeric nature of this protein. A more accurate determination of the molecular mass of PoSl was performed by MALDI-MS: a broad peak centered at 75,163 Da was obtained. The isoelectric point of the purified PoSl is 4.5.

View larger version (12K): [in this window] [in a new window]   FIG. 1.   Sequence of a tryptic peptide from P. ostreatus PoSl protease. The sequence has been aligned with those of other proteases belonging to the subtilase family (S8). An asterisk indicates the Asp residue present in the catalytic triad.

Catalytic properties. The activity of PoSl increased in the presence of CaCl₂ in the assay mixture at concentrations up to 2 mM and then was nearly constant at concentrations up to 10 mM, giving rise to a twofold increase in activity. Like calcium, Mn²⁺ similarly influenced proteolytic activity, while Zn²⁺and Mg² affected the activity to a lesser extent.

View larger version (10K): [in this window] [in a new window]   FIG. 2.   Comparison of cleavage sites of PoSl and other serine proteases in cleavage sites on oxidized bovine insulin B chain. Solid and broken arrows indicate preferential and secondary cleavage sites, respectively.

Autocatalytic assay. In order to evaluate the autocatalytic activity of PoSl, the enzyme was incubated at pH 8.5 at 37°C. The residual activities after 1, 3, and 18 h were 86, 25, and 0%, respectively. The recoveries of undigested PoSl, determined by HPLC analysis after 1, 3, and 18 h of incubation, were 72, 40, and 0%, respectively. These values are similar to those measured in an analogous reference experiment performed with chymotrypsin. Furthermore, calcium protects PoSl against autolysis; in fact, when incubation was performed in the presence of 10 mM CaCl₂, residual activities were 100 and 52% after 1 and 3 h, respectively.

Effect of inhibitors. The effect of various compounds on the ability of PoSl to hydrolyze SucAAPFpNA is shown in Table 3. PoSl activity was almost completely inhibited by antipain, chymostatin, PMSF, 3,4-dichloroisocoumarin, and 4-amidinophenyl-methanesulfonyl fluoride at the indicated concentrations (Table 3). Partial inhibition was observed using aprotinin, another serine protease inhibitor. Among the heavy metal ions tested, only Cu²⁺ caused a slight inhibition. None of the thiol-blocking reagents or metal-chelating agents affected the protease activity. These results, together with those described above, strongly suggest that PoSl belongs to the serine protease family.

Effect of PoSl on P. ostreatus laccase isoenzymes. The effect of PoSl on some P. ostreatus laccase isoenzymes, namely, POXC (17), POXA1b (12), and POXA3 (data to be published), was analyzed.

View larger version (43K): [in this window] [in a new window]   FIG. 3.   Zymogram of laccase isoenzymes incubated with purified PoSl protease. Fifty microliters of 2-day-old filtered culture broth that had been treated with PMSF (1 mM) was incubated with PoSl (0.01 nmol), and aliquots of equal volumes, withdrawn at different times, were loaded onto a native PAGE gel. Lane 1, control (sample incubated for 18 h in the absence of PoSl); lanes 2 to 4, samples incubated for 1, 3 and 18 h, respectively.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Proteolytic enzymes can play different roles in the physiology of ligninolytic fungi, such as activation of zymogenic enzymes or release of enzymes from the fungal cell walls. It has been previously reported (4, 8) that, in Phanerochaete chrysosporium under ligninolytic conditions, the turnover of lignin peroxidases is due in part to enzyme proteolysis, and specific proteases associated with conditions which promote lignin degradation have been identified. To the best of our knowledge, no relationship between manganese peroxidase and protease activity has been evidenced.

P. ostreatus produces several laccase isoenzymes putatively involved in lignin degradation (11, 12, 17). Recently, we demonstrated that the amounts of these isoenzymes increase substantially in copper-supplemented cultures and the presence of proteases secreted into the copper-containing culture medium specifically affects at least one of the known laccase isoenzymes (16). In order to better clarify the relationship between laccases and proteases in P. ostreatus and the role of these extracellular proteases, we have purified and characterized a protease (PoSl) from the fungal culture broth. PoSl is a monomeric glycoprotein with a molecular mass of 75 kDa, which is larger than that determined for most fungal proteases (5, 7, 9, 15, 19, 22).

This protease displays an optimum pH in the alkaline range, and its ability to release azo dye from Azocoll suggests that it is an endoprotease; moreover, the inhibitory profile indicates that PoSl is a serine protease. The sequences of the N terminus and of three tryptic peptides have been determined. The homology of one internal peptide with the conserved sequence around the Asp residue of the catalytic triad in the subtilase family (S8) suggests that PoSl is a subtilisin-like protein. This hypothesis is further supported by the finding that PoSl hydrolysis sites of the insulin B chain match those of subtilisin.

PoSl activity is positively affected by calcium; calcium ions, in fact, have a significant effect upon K_m values for two of the substrates tested (k_cat is almost unaffected), which can reflect an induced structural change in the substrate recognition site region. Furthermore, Ca²⁺ binding slows PoSl autolysis, triggering the protein to form a more compact structure. Both of these effects have been observed previously for subtilisin-like and other serine proteases (1, 10, 20).

The kinetic and structural characteristics of PoSl define it as a novel subtilisin-like protease that is different from the already known proteases from white rot fungi.

Recent reports on laccase isoenzymes secreted by P. ostreatus indicated an involvement of extracellular proteases in the degradation of POXA1b isoenzyme (16). In the present study, the effect of PoSl on laccase isoenzymes has been investigated. Results show that PoSl is actually involved in degrading POXA1b. In fact, POXA1b was unaffected in the PMSF-treated control (Fig. 3), while it was degraded in the presence of PoSl. On the other hand, no effect of PoSl on purified POXA1b was observed. These results suggest that PoSl plays a role in POXA1b degradation through either (i) a cascade activation mechanism of other proteases present in the culture broth or (ii) proteolysis of POXA1b leading to a polypeptide moiety, which is a better substrate for other proteases. A more significant effect of PoSl on laccase isoenzymes is the activation of POXA3. This effect can be due to PoSl-dependent POXA3 proteolysis generation of, in this case, a more active isoform(s). On the other hand, the possibility cannot be excluded that the observed activation could also be due to the PoSl-mediated hydrolysis of a polypeptide type of inhibitor. Further investigations are needed to discriminate between these two hypotheses.

No effect on POX isoenzymes was detectable when subtilisin was used instead of the fungal protease, although its specificity is actually very similar to that of PoSl. However, the possibility cannot be excluded that some differences in the specificities of these two proteases could be observable under different experimental conditions or when specific target proteins are used.

Reported results allow the conclusion that P. ostreatus extracellular proteases, besides their nutritional role, may be involved in the regulation of laccase activity by degrading or activating different isoenzymes.