Characterization of ScPrI, a small serine protease, from mycelia of Schizophyllum commune

726

Mycol. Res. 104 (6) : 726–731 (June 2000). Printed in the United Kingdom.

Characterization of ScPrI, a small serine protease, from mycelia of Schizophyllum commune

Jamie M. JOHNSTON, Erica R. RAMOS, Robert E. BILBREY, Allen C. GATHMAN and Walt W. LILLY Department of Biology, Southeast Missouri State University, Cape Girardeau, MO 63701, USA. E-mail : wlilly!biology.semo.edu Received 26 March 1999 ; accepted 17 August 1999.

Schizophyllum commune produces a variety of mycelial proteolytic enzymes. The specific functions of many of these enzymes are unknown, but several have elevated activity when the mycelium is grown in nitrogen-limiting conditions, suggesting a role in mycelial autolysis. We have purified one of these nitrogen-limitation induced enzymes, a small serine protease, ScPrI, from S. commune mycelial extracts. ScPrI has an apparent molecular mass of 22 kDa and is active against classical substrates for chymotrypsin and subtilisin proteases. The pH optimum for activity is neutral to slightly alkaline and the protein denatures above 50 mC. The enzyme is inhibited by PMSF, TPCK and chymostatin, and it shows little dependence on metal ions. Hydrolysis of oxidized insulin B-chain peptide by ScPrI demonstrated cleavage following aromatic amino acids and leucine. Kinetic analysis of hydrolysis of Nsuccinyl-AAPF-pNA and N-succinyl-AAPL-pNA revealed similar Kms for both substrates but the Vmax was nearly 3-fold higher for the substrate with phenylalanine in the P1 position.

INTRODUCTION Schizophyllum commune is a widely distributed basidiomycete that decays wood. Wood is a poor source of nitrogencontaining molecules and most data suggest that mycelial growth on wood is a nitrogen-limited process (Merrill & Cowling 1966, Levi & Cowling 1969, Park 1976). Nitrogenlimited growth of S. commune in plate culture results in markedly reduced biomass accumulation, but radial growth is maintained at a rate equal to that of cultures grown in nitrogen replete media (Sessoms & Lilly 1986). This sustained radial growth is supported by autolysis of older mycelia and subsequent translocation of nitrogen-containing molecules to the hyphal apices (Lilly, Wallweber & Higgins 1991). Mycelial proteins, upon hydrolysis, provide an abundant source of nitrogen for recycling. The mobilisation of this resource during autolysis is dependent upon a complex system of proteases. In S. commune this system comprises a ubiquitinmediated system in the cytoplasm (Higgins & Lilly 1993) in addition to a variety of endo- and exo-peptidases (Lilly et al. 1994). Of the latter group, two have been studied in detail. Aminopeptidase F is a novel enzyme that cleaves phenylalanine with high specificity from the amino terminus of proteins and peptides (Bilbrey et al. 1996). The role of this unique protease is unknown, but its activity is slightly increased during nitrogen limitation. The metalloendoprotease ScPrB is a Zn#+-dependent enzyme and is thought to be a primary protease involved in mycelial autolysis during nitrogen limited growth. Increased activity of ScPrB during

nitrogen depletion is localised to subapical regions of the mycelium (Gordon & Lilly 1995) and the enzyme appears to be vacuolar (Inselman, Gathman & Lilly 1999). Less is known about the complement of mycelial serine proteases produced by S. commune. One of these, ScPrA, is highly active but has been difficult to purify. In this study we have characterized ScPrI, the protease that migrates most rapidly in native gelatin containing polyacrylamide gels (Lilly, Higgins & Wallweber 1990). It is a serine protease with enzymatic characteristics similar to chymotrypsin and has substantially elevated activity during nitrogen limitation. MATERIALS AND METHODS Fungus and culturing methods Schizophyllum commune homokaryotic strain 4–40 (A43\B43) was grown as described in Bilbrey et al. (1996), except that -asparagine was used as the nitrogen source throughout. Briefly, 3i3 mm squares of mycelia taken from the margin of membrane-plate grown stock cultures were used to inoculate fresh minimal medium plates. The kinetics of N-succinyl-AlaAla-Pro-Phe-p-nitroanilide (iAAPF-pNA) hydolysing activity in plate culture were examined by the method of Gordon & Lilly (1995). After 96 h, the mycelia from two plates were harvested and macerated in 50 ml of liquid minimal medium using a Waring blender. Ten ml of this macerate was used to inoculate 50 ml of liquid minimal medium. After 2–3 d growth with rotary shaking at 200 rpm, the entire culture was grown

Downloaded from https://www.cambridge.org/core. Lund University Libraries, on 29 Sep 2019 at 04:42:59, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0953756299001793

J. M. Johnston and others

Purification of ScPrI ScPrI is reasonably stable for short periods. Using the small scale purification protocol outlined below, chromotographic procedures were carried out at 21m ; extract preparation, centrifugations and electrophoretic separations were done at 4m. Freshly thawed mycelia was suspended in 10 m Tris-HCl buffer, pH 7 (1 ml buffer for 1 g .. fungus) and homogenised for 10 min at high speed with a Kunkel Ultra-Turox tissue homogeniser. The homogenate was clarified by centrifugation at 10 000 g for 30 min and the supernatant frozen at k20m. After thawing, the homogenate was centrifuged again at 10 000 g for 15 min to remove insoluble material. Four ml of this crude extract was applied to a 1 ml Econo Pac2 (BioRad) ion-exchange cartridge containing MacroPrep Q resin equilibrated with 0n01  Tris-HCl, pH 7. After washing with 5 vol. of equilibration buffer, the column was eluted with a 25 ml linear gradient of 0 to 1  NaCl in 0n01  Tris-HCl, pH 7. One ml fractions were collected. Fractions containing activity against xAAPF-pNA were pooled and desalted into 1 m TrisHCl, pH 7, using a BioGel 10 DG column. The desalted pool (3–4 ml) was then applied to a 1 ml ceramic hydroxyapatite (CHT-II) column previously equilibrated with 10 m Naphosphate buffer, pH 6n8. The column was eluted with a 25 ml, 10 m to 400 m linear gradient of Na-phosphate buffer, pH 6n8. Two peaks of activity against xAAPF-pNA were resolved. The early peak corresponded to ScPrI. For some experiments the desalted ion-exchange pool was subjected to preparative IEF using the Rotofor system (BioRad). The focusing solution was 2 % (v\v) pH 3–10 ampholytes (BioRad), 11 % (v\v) glycerol in deionised water. The solution was pre-focused for 1 h, sample added, and focused for 4n5 h at 12 W constant current at 4m (Bilbrey et al. 1996). Determination of apparent molecular mass CHT-II purified ScPrI was chromatographed on a 1n5 cmi 50 cm Sephacryl S-200 column equilibrated in 0n1  Tris-HCl buffer, pH 7. Relative molecular weight was determined by comparing the Ve\Vo of ScPrI to that of these standards : thyroglobin, Mr l 670 000 ; gamma globulin, Mr l 158 000 ; ovalbumin, Mr l 44 000 ; myoglobin, Mr l 17 000 ; and vitamin B , Mr l 1350. Analytical SDS PAGE was routinely "# performed in 10 % T, mini-gels according to standard protocols (Laemmli 1970). Gels were stained for total protein (Silver Stain Plus, BioRad) and glycosylation (Immuno-Blot Kit, BioRad). Protein and enzyme assays Protein in samples was determined using the Bradford (1976) method with bovine serum albumin as the standard. ScPrI activity was determined by measuring the rate of hydrolysis

of xAAPF-pNA in 50 m Tris-HCl buffer, pH 7n5. Routine assays contained 900 µl bufferj50 µl 20 m xAAPF-pNAj 50 µl sample. Reactions were run at 30m with continuous shaking and A was determined spectrophotometrically. %#! One unit of ScPrI activity is defined as the amount of enzyme needed to release 1 µmol of p-nitroanilide min−" at 30m. Activity against other pNA substrates was determined in a similar manner. For inhibitor and cofactor studies, the assay was modified by reducing the amount of buffer to 890 µl and appropriate concentrations of inhibitor were added. Temperature stability was determined by incubating the standard reaction mix with out substrate for 30 min followed by addition of substrate and subsequent assay at the prescribed temperature. pH optimum was determined using the standard assay method with the substitution of 50 m citrate buffers for pH values 4–6. Tris-HCl buffer was used for pH 7–9. Data presented are means of replicate assays. The range around the mean was typically less than 15 %. Determination of cleavage specificity. Oxidised insulin B-chain was digested in a reaction mixture containing 2 nmol insulin in 50 m Tris-HCl buffer, pH 7n5. After overnight incubation at 25m, the samples were dried in a speed vac and subjected to MALDI-TOF spectroscopy (performed by Macromolecular Resources, Fort Collins CO, U.S.A.). Spectra obtained by MALDI-TOF were compared to those produced by hypothetical digestion of these substrate peptides by several common proteases (Protein Prospector, http :\\prospector.ucsf.edu\). RESULTS Purification of ScPrI N-succinyl-ala-ala-pro-phe-p-NA is a substrate commonly used to measure the activity of chymotrypsin-like and 18 xAAPF-pNA hydrolysing activity units (mg protein)–1

for an additional 2 d with rotary shaking at 200 rpm. Mycelia were harvested by filtration through cheesecloth, exhaustively washed with glass distilled water to remove extracellular polysaccharide slime, and frozen at k85 mC until used.

727

Min

16

M01

14 12 10 8 6 4 2 0

0

10

20 30 40 Time post-transfer (h)

50

60

Fig. 1. Specific activity of enzymes hydrolyzing N-succinyl-AAPFpNA from crude extracts of membrane plate grown S. commune homokaryotic strain 4-40. Mycelia were grown 4 d on minimal medium and transferred for the given times to either fresh minimal (Min) medium or to minimal medium containing 0n01 g l−" asparagine (M01). Data are means of replicate assays. Bars represent the range.

Downloaded from https://www.cambridge.org/core. Lund University Libraries, on 29 Sep 2019 at 04:42:59, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0953756299001793

728

10

450

120

9

400

8

100

350 300

6

250

5

200

4

150

3 2

100

80 60 40 20 0

50

1 0

Maximum activity (%)

7

4 Na-phosphate (mm)

xAAPF-pNA hydrolysing activity (U)

S. commune serine protease, ScPrI

1

6

11 16 Fraction

21

26

0

0

30

40

50

60

70

120 5 100 Maximum activity (%)

A

20

pH

Fig. 2. Fractionation of N-succinyl-AAPF-pNA hydrolysing activities by ceramic hydroxyapatite chromatography. - - l enzyme activity ; —— l Na-phosphate (m).

ScPr series A B

10

B

C D

80 60 40 20 0

E F G H

0

10

20 30 40 50 Temperature (°C)

60

70

Figs 4–5. Effect of pH on S. commune protease ScPrI. pH 4–6 buffer l 50 m citrate, pH 7–9 buffer l 50 m Tris-HCl. Fig. 5. Temperature stability of ScPrI. Samples incubated 30 min at temperature prior to addition of substrates. Assays run at indicated temperatures. Data are means and ranges of replicate assays.

I

Fig. 3. Native gelatin-containing PAGE of purified S. commune protease ScPrI. Lane A, ScPrI. Lane B, ScPrI incubated 5 min with 1 m PMSF prior to gel loading. ScPrx Series modelled after Lilly et al. 1994.

subtilisin-like proteases. Depriving plate-grown S. commune mycelia of a nitrogen source brings about an increase in the activity of intra-mycelial enzymes that hydrolyze this substrate (Fig. 1). Based on differential inhibition by 1 m PMSF and 1 m 1,10-phenanthroline, about 65 % of this activity can be attributed to serine proteases and the remainder to metalloproteases (data not shown). Partial purification of one of the enzymes responsible for hydrolysis of xAAPF-pNA was achieved by using a two step chromatographic procedure. Crude extract from liquid-grown cultures was applied directly to an ion-exchange MacroPrep Q column and the column was eluted with a linear NaCl gradient. The bulk of activity eluted

between 0n35  and 0n45  NaCl. The fractions containing xAAPF-pNA hydrolysing activity were pooled, desalted and applied to a ceramic hydroxyapatite column. This column was eluted with a linear phosphate gradient resulting in two peaks of activity (Fig. 2). The earlier peak was found to contain activity against xAAPF-pNA that could be completely inhibited by PMSF. The purified activity was identified as ScPrI by comparison of its migration in native gelatincontaining PAGE, to the ScPrx series previously described (Lilly et al. 1994) (Fig. 3). The later peak contained much less total activity and consisted of proteases of both the serine and metalloprotease classes (Johnston, unpublished). The results of this purification procedures are shown in Table 1. Properties of ScPrI Purified ScPrI was found to have a native molecular weight of 22 000 kDa determined by size exclusion chromatography.

Table 1. Purification of serine protease ScPrI from S. commune.

Step

Vol.

Total activity (U")

Total protein (mg)

Specific activity (U mg−" protein)

Yield (%)

Purification (-fold)

Crude extract Macroprep Q Hydroxyapatite

4 4 3

48n3 40n3 37n3

14n8 8n04 0n15

3n26 5n01 248n09

— 83 77

1 1n5 76

" Unit l µmol XAAPFpNA hydrolysed (min)−". Downloaded from https://www.cambridge.org/core. Lund University Libraries, on 29 Sep 2019 at 04:42:59, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0953756299001793

J. M. Johnston and others

729

Table 2. Hydrolysis of p-nitroanilide substrates by S. commune protease ScPrI. Relative rate" N-succinyl-AlaAlaProPhe-pNa N-succinyl-AlaAlaProLeu-pNA Phe-p-NA Pro-p-NA N-succinyl-TyrLeuVal-pNA Gly-pNA GlyPhe-pNA GlyArg-pNA GlyPro-pNA N-succinyl-Phe-pNA

100 15 6 2 1 1 1 0n5 0n4 0

" Rate of hydrolysis of N-succinyl-AlaAlaProPhe-pNA l 3n75 µmol (min)−".

Table 3. Effects of mechanistic inhibitors on activity of S. commune ScPrI.

None PMSF Chymostatin TPCK TLCK APMSF 1, 10 Phenanthroline EDTA DTT E-64 Pepstatin A Bestatin

Concentration ( µ)

Activity control (%)

na 2000 20 000 165 330 30 300 30 300 50 500 2000 20 000 2000 20 000 1000 10 000 2000 20 000 1 10 100 200

100" 2 0 2 2 68 60 104 111 108 96 94 51 86 117 100 105 100 87 106 107 110 110

" Assays performed with 720 mU of ScPrI. Abbreviations : PMSF l phenylmethylsulphonyl fluoride ; TPCK, tosyl phenylalanyl chloromethyl ketone ; TLCK, tosyl leucyl chloromethyl ketone ; APMSF, 4-(amidophenyl) methanesulphonyl fluoride ; EDTA, ethylenediamine tetraacetic acid ; DTT, dithiothreitol ; E-64, -trans-epoxysuccinyl-leucylamide-(4-guanidino)-butane.

Silver-stained SDS polyacrylamide gels (0n5 µg total protein load) showed a very faint band near this same molecular weight in addition to two or three contaminating proteins of similar intensity at higher molecular weights. Analysis with a BioRad glycosylation kit indicated that the 22 000 kDa protein was not glycosylated. SDS-gelatin gel analysis showed that while ScPrI was active, no other protease bands were present indicating that the contaminating bands were not proteases. Preparative fractionation in a Rotofor system showed the isoelectric point of ScPrI to be between 4n5 and 5n0. ScPrI was heat stable to at least 45m and had a broad pH optimum in the neutral to lower basic range (Figs 4–5). Of the p-nitroanilide substrates tested, ScPrI showed the greatest hydrolysis rate against xAAPF-pNA (Table 2). The

Fig. 6. Proteolytic fragments generated by digestion of oxidized insulin B-chain by S. commune protease ScPrI. C* l oxidised cysteine residue. Arrows l predicted cleavage sites of chymotrypsin ; dashed arrow l predicted chymotrypsin cleavage site NOT demonstrated for ScPrI.

substitution of leucine for phenylalanine in the P1 position of the substrate resulted in an 85 % loss of activity. The enzyme also showed very little activity against short peptides with phenylalanine in the P1 position. S. commune produces no trypsin-like activities so N-blocked substrates with arginine and lysine in the P1 position were not tested. Complete inhibition by PMSF, a mechanistic inhibitor for serine proteases, demonstrated unequivocally that ScPrI belongs to this class (Table 3). Potent inhibition by chymostatin narrows the classification to a chymotrypsin-like serine protease. PMSF and chymostatin also inhibit some cysteine proteases ; however, the lack of inhibition of ScPrI activity by the classspecific irreversible cysteine protease inhibitor E-64 excludes the possibility that ScPrI is a cysteine protease. In addition, TPCK, an inhibitor with relatively high specificity for chymotrypsin showed moderate inhibition of the enzyme at the concentrations tested. With the exception of very high concentrations of 1,10 phenanthroline, other class specific inhibitors showed little effect on ScPrI activity. The reason for the decreased activity caused by high 1,10-phenanthroline is not clear. It is interesting to note that high concentrations of the other chelator, EDTA, had the opposite effect as 1,10phenanthroline. The apparent effects of 1,10-phenanthroline and EDTA led to the examination of the role of divalent cations in ScPrI activity. There were minimal effects, and no activation of ScPrI by Ca#+, Mg#+, and Co#+ at concentrations of 10 m. One m Cu#+ inhibited activity by 55 % and 10 m Cu#+ reduced activity to near zero. Ten m Zn#+ inhibited activity by about 40 %. Most chymotrypsin enzymes cleave after amino acids with aromatic side chains or after leucine. The Km of ScPrI for the substrate xAAPF was found to be 0n22p0n07 m. Similar results (Km l 0n28p0n03 m) were obtained for the substrate xAAPL-pNA which has Leu in the P1 site. The Vmax for xAAPF-pNA, 3591 nKat (mg protein)−", however, was nearly three fold higher than for xAAPL-pNA, 1052 nKat (mg protein)−", explaining the apparent higher rate of hydrolysis of the former substrate. This cleavage specificity is supported by data from digestion of oxidised insulin B-chain peptide.

Downloaded from https://www.cambridge.org/core. Lund University Libraries, on 29 Sep 2019 at 04:42:59, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0953756299001793

S. commune serine protease, ScPrI Hypothetical limited digestion of this peptide with chymotrypsin yields 45 fragments. The larger of these fragments provided the most useful substrate specificity data. An actual digestion of oxidised insulin B-chain with ScPrI produced six fragments with masses above 1500 kDa corresponding to cleavage after phenylalanine, tyrosine, or leucine. In addition, several smaller fragments were obtained, showing similar cleavage sites. The alignment of fragments generated from ScPrI digest of insulin B-chain with the hypothetical chymotrypsin digest fragments demonstrates the overall cleavage site similarity between the two enzymes (Fig. 6). No fragment was found corresponding to cleavage after leucine in position 6, perhaps due to the presence of the oxidised cysteine in the P1h position.

730 protein. Inhibition with TPCK and chymostatin in addition to insulin B-chain cleavage patterns suggest a close alliance to chymotrypsin-like enzymes. The fungal subtilisins that have been characterised by insulin cleavage show additional cleavage after the fourth amino acid in the sequence, glutamine (Abraham, Chow & Breuil 1995). Fragments corresponding to this cleavage point were never observed in insulin B-chain digests with ScPrI. In addition, ScPrI cleaves after the Nterminal phenylalanine, and at all leucines except leu' unlike the subtilisins from Ophiostoma picae and Aspergillus oryzae (Abraham, Chow & Breuil 1995). ACKNOWLEDGEMENT This work was supported by a grant from the National Science Foundation (MCB 96-31340) to WWL and ACG.

DISCUSSION ScPrI is a rapidly migrating gelatinase that is detected exclusively when mycelia of S. commune are grown under conditions of nitrogen deprivation (Lilly et al. 1996). The activity of this gelatinase was found to be inhibited by PMSF, indicating that it is a serine protease. The two-step chromatographic purification for ScPrI is a rapid procedure for obtaining small quantities of the enzyme with reasonable yield and purity. This procedure differs from those previously published (Wallweber & Lilly 1992, Bilbrey et al. 1996), a chromatographic separation is used as a first step. The copious amounts of a polysaccharide slime in extracts has a tendency to rapidly clog columns. Using MacroPrep Q columns allows for higher pressures and flow rates, making it possible to run an initial ion exchange separation directly on crude extracts. It is necessary to completely clean the column with 1  NaOH after each run because the capacity is reduced substantially by non-specific binding of the slime to the column. While several proteolytic activities co-purify in the first chromatographic step, ScPrI is isolated from all other proteases by hydroxyapatite chromatography. Purified ScPrI has a molecular weight of around 22 000 kDa and a pI near 5. These are typical for many classes of serine proteases, including trypsin, chymotrypsin, elastase and subtilisin (Lesk & Fordham 1996, North 1982). The pH optimum and temperature denaturation profiles for ScPrI are also similar to many enzymes in these classes (Dunn 1989). Traditionally, microbial serine proteases have been considered to be members of the subtilisin class of enzymes and sequence comparisons have generally borne this out (Abraham & Breuil 1996, Joshi, St Leger & Roberts 1997). Unfortunately, the few serine proteases from filamentous fungi that have been characterized are nearly all extracellular enzymes rather than mycelial (Rao et al. 1998). ScPrI shares some characteristics with a serine protease isolated from Agaricus bisporus basidiocarps (Burton et al. 1993), including small size (ca 27 kDa), pH optimum and kp-nitroanilide substrate hydolysis specificity. The A. bisporus enzyme has a much higher pI than ScPrI, however, and would not cleave short peptides. Nterminal sequencing of the A. bisporus protease showed it had homology to proteinase K and to other subtilisin-like proteases. Whether ScPrI is a subtilisin-like protease will only be determined when sequence information is available for this

REFERENCES Abraham, L. D. & Breuil, C. (1996) Isolation and characterization of a subtilisin-like serine protease secreted by the sap-staining fungus Ophiostoma piceae. Enzyme and Microbial Technology 18 : 133–140. Abraham, L. D., Chow, D. T. & Breuil, C. (1995) Characterization of cleavage specificity of a subtilisin-like serine protease from Ophiostoma piceae by liquid chromatography\mass spectrometry and tandem MS. FEBS Letters 374 : 208–210. Bilbrey, R. E., Penheiter, A. R., Gathman, A. C. & Lilly, W. W. (1996) Characterization of a novel phenylalanine-specific aminopeptidase from the basidiomycete, Schizophyllum commune. Mycological Research 100 : 462–466. Burton, K. S., Wood, D. A., Thurston, C. F. & Barker, P. J. (1993) Purification and characterization of a serine proteinase from senescent sporophores of the commercial mushroom Agaricus bisporus. Journal of General Microbiology 139 : 1379–1386. Dunn, B. M. (1989) Determination of protease mechanism. In Proteolytic Enzymes, a Practical Approach (R. J. Benyon & J. S. Bond, eds) : 57–82. IRL Press, Oxford. Gordon, L. J. & Lilly, W. W. (1995) Quantitative analysis of Schizophyllum commune metalloprotease, ScPrB activity in SDS-Gelatin PAGE reveals differential mycelial localization of nitrogen-limitation induced autolysis. Current Microbiology 30 : 337–343. Higgins, S. M. & Lilly, W. W. (1993) Multiple responses to heat stress by the basidiomycete fungus Schizophyllum commune. Current Microbiology 26 : 123–127. Inselman, A. L., Gathman, A. C. & Lilly, W. W. (1999) Two fluorescent markers identify the vacuolar system of Schizophyllum commune. Current Microbiology 38 : 295–299. Joshi, L., St Leger, R. J. & Roberts, D. W. (1997) Isolation of a cDNA encoding a novel subtilisin-like protease (Pr1B) from the entopathogenic fungus, Metarhisium anisoplie using differential display-RT-PCR. Gene 197 : 1–8. Lesk, A. M. & Fordham, W. D. (1996) Conservation and variability in the structures of serine proteases of the chymotrypsin family. Journal of Molecular Biology 258 : 501–537. Levi, M. P. & Cowling, E. B. (1969) Role of nitrogen in wood deterioration. VII. Physiological adaptation of wood-destroying and other fungi to substrates deficient in nitrogen. Phytopathology 59 : 460–468. Lilly, W. W., Bilbrey, R. E., Williams, B. L., Loos, L. S., Venable, D. F. & Higgins, S. M. (1994) Partial characterization of the proteolytic system of Schizophyllum commune. Mycologia 86 : 564–570. Lilly, W. W., Higgins, S. M. & Wallweber, G. J. (1990) Electrophoretic detection of multiple proteases from Schizophyllum commune. Mycologia 82 : 505–508. Lilly, W. W., Wallweber, G. J. & Higgins, S. M. (1991) Proteolysis and amino acid recycling during nitrogen deprivation in Schizophyllum commune. Current Microbiology 23 : 27–32. Merrill, W. & Cowling, E. B. (1966) Role of nitrogen in wood deterioration : amount and distribution of nitrogen in tree stems. Canadian Journal of Botany 44 : 1555–1579.

Downloaded from https://www.cambridge.org/core. Lund University Libraries, on 29 Sep 2019 at 04:42:59, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0953756299001793

J. M. Johnston and others North, M. J. (1982) Comparative biochemistry of the proteinases of eucaryotic microorganisms. Microbiological Reviews 46 : 308–340. Park, D. (1976) Carbon and nitrogen levels as factors influencing fungal decomposers. In The Role of Terrestrial and Aquatic Organisms in Decomposition Processes (J. M. Anderson & A. Macfayden, eds) : 41–59. Blackwell, Oxford. Rao, M. B., Tanksdale, A. M., Ghatge, M. S. & Deshpande, V. V. (1998) Molecular and biotechnological aspects of microbrial proteases. Microbiology and Molecular Biology Reviews 62 : 597–635.

731 Sessoms, D. B. & Lilly, W. W. (1986) Derepressible proteolytic activity in homokaryotic hyphae of Schizophyllum commune. Experimental Mycology 10 : 294–300. Wallweber, G. J. & Lilly, W. W. (1992) Purification and characterization of the two constitutively produced acid phosphatase isozymes from Schizophyllum commune. Mycological Research 96 : 792–797. Corresponding Editor : D. A. Wood

Downloaded from https://www.cambridge.org/core. Lund University Libraries, on 29 Sep 2019 at 04:42:59, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0953756299001793