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Protease production by the thermophilic fungus Thermomyces lanuginosus
Mycol. Res. 101 (1), 18–22 (1997)
18
Printed in Great Britain
Protease production by the thermophilic fungus Thermomyces lanuginosus
D U O - C H U A N L I, Y I - J U N Y A N G A N D C H O N G -Y A O S H E N Department of Plant Pathology, China Agricultural University, Beijing 100094, P.R. China
A total of 500 strains of the thermophilic fungus Thermomyces lanuginosus were screened for their ability to produce proteases. A selected strain, T. lanuginosus P , showed high enzymatic activity for proteases. The maximal yield of proteases was obtained with "$% 4 % casein, 4 % glucose and 4 % yeast extract in submerged culture. The protease system was shown to contain at least two protease isoenzymes by SDS–PAGE with gels containing gelatin as protease substrate. The effects of inhibitors imply that the two enzymes appear to be serine proteases. The crude proteases of T. lanuginosus P displayed unusual properties. The proteases were shown to "$% be one of the most thermostable proteases so far isolated in fungi. The proteases were 100 % stable at 50 °C. The half-life at 60 and 70° was approximately 160 min and 60 min, respectively. Temperature optimum of activity was 70°. The crude proteases were stable over a broad pH range (4–11). Optimum pH of activity was at 5±0 and 9±0.
Proteases are an important group of enzymes both physiologically and commercially. Microbial proteases dominate commercial applications (Ward, 1983 ; Outtrup & Boyce, 1990). A major requirement for commercial enzymes is thermal stability, because thermal denaturation is a common cause of enzyme inactivation (Kristjansson, 1989). In recent years, there has been increasing interest in proteases from thermophiles, which were expected to produce thermostable proteases. From known reports, most of the research has been on proteases from thermophilic bacteria of the genera Bacillus and Thermus, and reports from thermophilic fungi are limited (Ong & Gaucher, 1973, Aunstrup, 1980 ; Ward, 1983 ; Kristjansson, 1989 ; Outtrup & Boyce, 1990). The thermophilic Thermomyces lanuginosus Tsikl. (formerly Humicola lanuginosa ; Domsch, Gams & Anderson, 1980), has been shown to produce proteases (Ong & Gaucher, 1973), but detailed information is sparse. In this paper we report on the production, identification and partial characterization of the crude proteases by T. lanuginosus.
previously (Cooney & Emerson, 1964). Identification was conducted mainly according to the taxonomical methods of Cooney & Emerson (1964) and Domsch et al. (1980). Selection. All strains isolated were maintained on YpSs agar. A strain P with high protease activity was selected by "$% inoculating isolates onto yeast extract agar plates with 1 % skim milk. Growth conditions. T. lanuginosus was grown in shake cultures at 50 °C on a modified medium from Ong & Gaucher (1973) containing the following (g l−") : varying amounts of casein, glucose and yeast extract (Table 1) ; 1 g Table 1. Effect of concentration of casein, glucose, and yeast extract on protease production by T. lanuginosus. The crude proteases were from 12d-old submerged cultures of T. lanuginosus at 50° Protease activity (µ ml−")*
MATERIALS AND METHODS
Treatment medium
pH 5±0
pH 9±0
Materials. Casein, gelatin, iodoacetamide, acrylamide, bisacrylamide, bromophenol blue, Tris, glycine and tyrosine were from Sigma, PMSF, EDTA, Coomassie brilliant R , SDS, #&! TEMED, Triton X-100 and ammonium persulphate were from Serva, Yeast extract was from Difco, and soluble starch was from Merck. Other reagents were of analytic grade and were obtained from Beijing Chemical Reagent Corporation (Beijing, China).
4 % casein, 0±6 % glucose, 0±4 % yeast extract 4 % casein, 0±4 % glucose, 0±4 % yeast extract 4 % casein, 0±2 % glucose, 0±4 % yeast extract 4 % casein, 0±4 % yeast extract 4 % casein, 0±4 % glucose, 0±6 % yeast extract 4 % casein, 0±4 % glucose, 0±2 % yeast extract 4 % casein, 0±4 % glucose 4 % soluble starch, 0±4 % yeast extract 4 % glucose, 0±4 % yeast extract 2 % casein, 0±4 % glucose, 0±4 % yeast extract 6 % casein, 0±4 % glucose, 0±4 % yeast extract 0±4 % glucose, 0±4 % yeast extract
8±0³0±3 14±0³0±2 10±5³0±6 9±0³0±6 12±8³0±3 11±2³0±5 4±5³0±6 3±0³0±5 3±0³0±3 10±0³0±7 6±0³0±2 1±7³0±3
8±6³0±1 12±3³0±1 11±0³0±3 7±2³0±2 10±0³0±2 9±5³0±1 3±6³0±2 3±5³0±2 4±0³0±9 7±0³0±4 4±3³0±7 2±2³0±6
Isolation and identification. Strains of T. lanuginosus were isolated from compost, dung and soil on Emerson’s YpSs (Yeast Extract Soluble Starch) agar by a method described
* Values are the mean³... of two experiments with three replicates each.
D.-C. Li, Y.-J. Yang and C.-Y. Shen K HPO \3H O ; 0±5 g MgSO \7H O ; 0±1 ml of Vogel’s # % # % # trace element solution (Vogel, 1956). The pH was adjusted to 7±4 with 1 N NaOH. After different times, the mycelium was filtered off, then the culture filtrate was centrifuged at 10 000 g for 30 min at 4°, and the resultant supernatant was dialysed overnight against three changes of water. The dialysed sample of crude proteases was stored at 0°. Enzyme assays. The protease activity of the crude proteases was determined by using the modified Rick’s method (Rick, 1974). The enzymes (0±5 ml) were added to 2±5 ml of 0±4 % casein in 0±2 Tris}HCl (pH 9±0) or 0±2 CH COOH} $ CH COONa (pH 5±0) buffer and incubated at 50° for $ 60 min. Two ml 10 % TCA (Trichloroacetic acid) was then added, and after standing at room temperature for 30 min, the solution was filtered. To 1 ml of filtrate, 5 ml of water was added, the optical density (O.D.) at 280 nm was determined spectrophotometrically in UV-160A (Shimadzu). The blank used was prepared by adding 10 % TCA to the enzyme before addition of casein. One unit of protease activity was defined as the amount of enzyme producing 1 µg tyrosine min−" under assay conditions. Electrophoretic procedures. SDS–PAGE was used to establish the number of electrophoretically distinct proteases, to follow changes in protease profiles during growth, and to identify the protease isoenzymes of T. lanuginosus. Electrophoretic separation of proteases was performed in 4 % stacking}10 % separation gel. The separation gel composition was as follows : acrylamide (30 %) and bisacrylamide (1 %) in distilled water 3±3 ml ; 1±5 Tris}HCl (pH 8±8), containing 0±4 % SDS 2±25 ml ; gelatin (1 %) in distilled water 0±9 ml ; ammonium persulphate (10 %) in water 0±02 ml ; TEMED 0±01 ml ; water 2±45 ml. The stacking gel composition was as follows : acrylamide (30 %) and bisacrylamide (1 %) in distilled water 0±2 ml ; 0±5 Tris}HCl (pH 6±8), containing 0±4 % SDS 0±25 ml ; ammonium persulphate (10 %) in water 0±02 ml ; TEMED 0±01 ml ; water 1±55 ml. Samples of equal volumes in sample buffer (0±1 Tris}HCl, pH 6±8, containing 2 % (w}v) SDS, 10 % (v}v) glycerol and bromophenol blue as front marker) were electrophoresed at 10 mA constant current at 4° for 3 h, using the buffer system described by Laemmli (1970). After separation proteins were renatured by washing the gels in 50 m Tris}HCl (pH 9±0) or 50 m CH COOH}CH$ COONa (pH 5±0), containing 5 % (v}v) Triton X-100 twice $ for 15 min at 4°, followed by one 15 min wash at 4° in the same buffer without Triton X-100. The gels were then incubated in 50 m Tris}HCl (pH 9±0) or 50 m CH COOH} $ CH COONa (pH 5±0) for 3 h at 50° to allow degradation of $ the gelatin. Gels were stained in 0±1 % (w}v) Coomassie brilliant blue G250 reagent in 45 % (v}v) methanol and 10 % (v}v) acetic acid and destained in 30 % (v}v) methanol and 10 % (v}v) acetic acid. For further identification different inhibitors specific for serine-proteases (phenylmethyl sulphonyl fluoride, PMSF), cystein-proteases (iodoacetamide), and metallo-proteases (ethylenediaminetetraacetic acid, EDTA) were incubated with the enzymes. PMSF (0±5 m) was added to the sample and incubated for 25 min on ice prior to electrophoresis.
19 Iodoacetamide (0±5 m) and EDTA (30 m) were added to the gel washes and were also present during gel incubation. Protease partial characterization. The influence of pH on protease activity was determined by incubating the enzyme with casein for 60 min at 50° in the presence of buffers of a wide pH range (pH 2–12). The buffers used were : 0±2 HCl}KCl (pH 2–3) ; 0±2 CH COOH}CH COONa $ $ (pH 4–5) ; 0±2 NaH PO }Na HPO (pH 6–7) ; 0±2 Tris} # % # % NaOH (pH 8–12). Activity was estimated as a percentage of the maximum. Temperature optimum was determined by incubating the proteases with casein and buffer for 60 min at different temperatures. Thermal stability of proteases was examined in the range 50–80° at pH 5±0 and pH 9±0. The proteases were incubated in buffers without addition of casein, and samples were removed at fixed time intervals and allowed to cool on ice before the residual activities were determined under standard conditions. The buffers used were 0±2 CH COOH} $ CH COONa (pH 5±0) and 0±2 Tris}HCl (pH 9±0). Activity $ was measured as a percentage of the maximum. The protease inhibitors, PMSF, iodoacetamide and EDTA, were used to determine the effects on protease activity. PMSF was dissolved in 100 % methanol ; EDTA and iodoacetamide were dissolved in distilled water. The enzymes were preincubated with each inhibitor at either pH 5±0 or 9±0 for 1 h at room temperature. The samples were subsequently added to the buffer with casein for 60 min at 50°. Control with distilled water and methanol was treated in the same way as the test samples. The buffer used was 0±2 CH COOH} $ CH COONa (pH 5±0) and 0±2 Tris}HCl (pH 9±0). $ RESULTS Isolation and identification of T. lanuginosus About 500 strains of T. lanuginosus were isolated and identified. The strains isolated were characterized by colonies on YpSs media reaching a diameter of 9 cm in 5 d at 50°, white, later becoming grey, and eventually black, with a pink to vinaceous pigment diffusing into the agar. Aleurioconidia arising on lateral stalk cells, singly, at first hyaline, then becoming dark brown, globose, 5–9 µm diam., thick-walled, verruculose. Good growth occurred at temperatures between 40 and 55°, the minimum was about 35°, the optimum 50° and the maximum about 60°. Selection of a high-yield protease strain of T. lanuginosus Following growth for 72 h on yeast extract agar plates with 1 % skim milk, a clearing zone was observed around the colonies, indicating protein degradation. Among 500 strains, P strain produced the widest clearing zone. The strain was "$% selected for further studies. Influence of the carbon and nitrogen on extracellular protease production Four percent (w}v) casein, 0±4 % (w}v) glucose, and 0±4 %
Production of proteases by Thermomyces lanuginosus
20
14
12
12
10
10
8
8
6
6
4
4
2
2
0
0 14
0
2
4 6 8 10 Time since inoculation (d)
12
100 Protease activity (%)
14
pH Dry weight (g ml–1)
Protease activity (U ml–1)
16
Time course of extracellular protease production The time course of extracellular protease production from submerged cultures grown on 4 % casein, 0±4 % glucose and 0±4 % yeast extract of T. lanuginosus at 50° is shown in Fig. 1. It is clear from these experiments that protease formation was associated with mycelial growth. Large increases in protease activity paralleled rapid increases in mycelial biomass. Protease activity in the medium reached a maximum at 12 d. At the same time the maximum dry weight was 5±1 gl−". The pH dropped slowly to 5±8 at 4 d followed by an increase to a maximum at 12 d. SDS–PAGE profiles and isoenzymes of proteases produced at various growth times The samples from different growth time were separated by SDS–PAGE into two proteolytic major bands active at pH 5±0 and pH 9±0 (Fig. 2). The isoenzymes were designated protease I and protease II, according to their electrophoretic mobility, with protease I having the lower mobility. 10
40
0
(w}v) yeast extract gave the highest yield of proteases (Table 1).
12
60
20
Fig. 1. Time course of growth and protease production by T. lanuginosus. The crude proteases were from submerged cultures grown on 4 % casein, 0±4 % glucose, and 0±4 % yeast extract of T. lanuginosus at 50° at different growth stages. Activity was assayed as in experimental procedure. +, activity (at pH 5±0) ; *, activity (at pH 9±0) ; E, dry weight ; _, pH. Values are the mean³... of two experiments with three replicates each. Error bars show ...
14
80
8
pH 9.0
6
4
2
2
4
6
8
10
12
14
pH
Fig. 3. Effect of pH on protease activity of T. lanuginosus. The crude proteases were from 12-d-old submerged cultures of T. lanuginosus grown on 4 % casein, 0±4 % glucose and 0±4 % yeast extract at 50°. Activity was estimated at pH 2–12 and 50° for 1 h. Values are the mean³... of two experiments with three replicates each. Error bars show ...
Protease I appeared at day 4, and reached a maximum at 12 d (Fig. 2). Protease II presented at 6 d and reached a maximum at 12 d. Clearly, the profiles observed in the gel (Fig. 2) are consistent with the changes in activity shown in Fig. 1. Partial characterization of T. lanuginosus P134 proteases The sample from 12 day old submerged cultures grown on 4 % casein exhibited optimal activity from 4±0 to 11±0. Outside this range, activity was lost rapidly. Optimum pH of the sample was at 5±0 and 9±0 (Fig. 3). Activity increased rapidly from 40° to a maximum at 70°, with rapid loss of activity at temperatures above 70° (Fig. 4). At 50° the proteases were 100 % stable (Fig. 5). The halflife of the proteases at 60 and 70° was approximately 160 min and 60 min, respectively, and at 80° the activity was lost within 5 min. Thermostability at pH 9±0 was similar (data not shown). The proteolytic activity of the sample was completely inhibited at acid and alkaline pH values by PMSF, and not inhibited by iodoacetamide and EDTA (Table 2). This indicates the presence of serine protease(s) within the sample, with little or no cysteine protease(s) and metalloprotease(s). 14
12
10
8
6
4
2
pH 5.0
Fig. 2. Patterns of Thermomyces lanuginosus proteases after different periods of growth in submerged cultures (2, 4, 6, 8, 10, 12, 14 d). After electrophoretic separation the gel was incubated remaining substrate was stained, and destained. The assays were carried out at 50 m CH COOH}CH COONa (pH 5±0) and 50 m Tris}HCl (pH 9±0). $ $
D.-C. Li, Y.-J. Yang and C.-Y. Shen
21 Table 2. Effect of inhibitors on protease activity of T. lanuginosus. The crude proteases were from 12-d-old submerged cultures grown on 4 % casein, 0±4 % glucose and 0±4 % yeast extract of T. lanuginosus at 50°. The crude proteases were pre-incubated with each inhibitor at either pH 5±0 or pH 9±0 for 1 h at room temperature
Protease activity (%)
100 80
Inhibition (as % of control)*
60
Inhibitor
pH 5±0
pH 9±0
40
PMSF (0±5 m) Iodoacetamide (0±5 m) EDTA (30 m)
100 (100³1±32) 0 (0±21³0±08) 0 (0±14³0±02)
100 (100³1±78) 0 (0±39³0±04) 0 (0±36³0±03)
* Values are the mean³... of two experiments with three replicates each.
20
0
20
40
60 80 Temperature (C)
100
120
Fig. 4. Effect of temperature on protease activity of T. lanuginosus. The crude proteases were from 12-d-old submerged cultures of T. lanuginosus grown on 4 % casein, 0±4 % glucose, and 0±4 % yeast extract at 50°. Activity was estimated at pH 5±0 (+) and pH 9±0 (E) at each temperature for 1 h. Values are the mean³... of two experiments with three replicates each. Error bars show ...
120
Protease activity (%)
100 80 60 40 20
0
50
100 150 Time (min)
200
250
Fig. 5. Thermal stability of proteases from T. lanuginosus. The crude proteases were from 12-d-old submerged cultures of T. lanuginosus grown on 4 % casein, 0±4 % glucose and 0±4 % yeast extract at 50°. The crude proteases were pre-incubated in buffers without addition of casein, and samples were removed at fixed time intervals and allowed to cool on ice before the residual activities were determined at pH 5±0 at 50° for 1 h. +, 50° ; *, 60° ; _, 70° ; E, 80°. Values are the mean³... of two experiments with three replicates each. Error bars show ...
Inclusion of the inhibitors iodoacetamide and EDTA in the protease gel assay did not appear to alter the enzyme pattern of the crude proteases at pH 5±0 and pH 9±0 (Fig. 6). In contrast, PMSF reduced the intensities of two protease main bands (protease I and protease II) at pH 5±0 and pH 9±0, indicating that protease I and protease II are serine proteases. DISCUSSION Regulation of extracellular protease production has been studied in a number of fungi and normally involves either
induction or repression}depression or a combination of both (Lasure, 1980 ; North, 1982 ; Kalisz, Wood & Moore, 1987 ; Farley & Ikasari, 1992). In this study, extracellular protease production by T. lanuginosus was induced by protein such as casein and not induced by starch. Higher concentrations of casein yielded lower amounts of enzyme, presumably because of degradation of casein to amino acids by enzyme in the medium, and subsequent repression of enzyme production by amino acids. Glucose repressed extracellular protease production, though a small amount of glucose increased protease production. Similar observations have been made previously (Ong & Gaucher, 1973). Protease production was also stimulated by yeast extract, but higher concentration of yeast extract diminished protease production. This is likely because yeast extract increased conidum germination and mycelial growth of T. lanuginosus (Haasum et al., 1991 ; Jensen et al., 1993), which increased protease production ; on the other hand, amino acids in yeast extract repressed protease production, which decreased protease production. Proteases are a complex group of enzymes varying greatly in their physicochemical and catalytic properties. They are generally classified by pH optimum and catalytic mechanism. On the basis of pH optimum, proteases are classified into acid, neutral, and alkaline proteases. Acid, neutral and alkaline proteases have pH optima of 2±0–5±0, 7±0–8±0 and 7±0–11±0, respectively. On the basis of catalytic mechanism, proteases are classified into serine proteases, cysteine proteases, aspartic proteases and metalloproteases, determined indirectly through reactivity towards inhibitors of particular amino acid residues in the active site region (North, 1982). With respect to pH optimum, T. lanuginosus P proteases had a very broad pH "$% optimum. There was little or no difference in properties between at pH 5±0 and at pH 9±0. Therefore, T. lanuginosus P proteases cannot be classified into acid, neutral or alkaline "$% proteases. With respect to inhibitor sensitivity, T. lanuginosus P proteases were inhibited by the serine inhibitor PMSF, "$% but not by thiol reagents such as iodoacetamide. Therefore, the proteases are classified as serine proteases. Ong & Gaucher (1973) reported that 20 and 80 % inhibition of extracellular proteases of T. lanuginosus was caused by DFP and pCMB, respectively. This suggests the presence of serine proteases, but it is not definite whether there were cysteine proteases, since many serine proteases in fungi are also inhibited by thiol reagents (North, 1982). In our experiments, the assays showed 100 % inhibition
Production of proteases by Thermomyces lanuginosus 1
2
3
22 4
1
2
3
GHOST IMAGE
GHOST IMAGE
pH 9.0
pH 5.0
4
Fig. 6. Effect of different inhibitors on Thermomyces lanuginosus proteases. Inhibitors of cysteine proteases (Iodoacetamide, 0±5 m ; lane 2) and metalloproteases (EDTA, 30 m ; lane 3) were added to the gel washing and incubation buffers. Inhibitor of serine proteases (PMSF, 0±5 m ; lane 4) was added to the samples before SDS–PAGE. Untreated proteases served as control (lane 1). The assays were carried out at 50 m CH COOH}CH COONa (5±0) and 50 m Tris}HCl (9±0). $ $
(Table 2) and the gels showed only partial inhibition (Fig. 6). There are two possible reasons for this : (i) sensitivity of the assays is lower than that of the gels ; (ii) reaction time of the assays is shorter than that of the gels. There is possibly not 100 % inhibition in the assays, but because reaction time is short and product formed is very low, the difference in activity is not detected. The proteases from T. lanuginosus P described in this "$% paper displayed a high thermal stability as compared with those of other proteases in fungi (Aunstrup, 1980). The proteases were proven to be some of the most thermostable so far isolated in fungi, though not as much as those exhibited by bacteria such as Bacillus amyloliquefaciens (Aunstrup, 1980), Sulfolobus solfataricus (Burlini et al., 1992), Desulfurococcus mucosus (Cowan et al., 1987), Sulfolobus acidocaldarius (Lin & Tang, 1990), and Pyrococcus furiosus (Connaris, Cowan, & Sharp, 1992). The half-life of the proteases at 70° was 60 min in the absence of Ca#+. To our knowledge, among fungal proteases only the extracellular serine protease from the thermophilic fungus Malbranchea pulchella var. sulfurea displays a comparable thermostability. The half-life of the serine protease at 73° was 7±5 min in the absence of added Ca#+ and increased to 110 min upon addition of Ca#+ (Ong & Gaucher, 1975). The molecular basis of thermostability is still poorly understood. The hydrophobic bonds, ionic interactions and disulphide bridges in the enzyme molecule may be involved in thermostability (Kristjansson, 1989). From the characteristics of T. lanuginosus P proteases, it "$% would appear that the proteases may be of potential value in industry. Further studies on proteases of T. lanuginosus P "$% are necessary. Purification of the proteases is in progress in our laboratory. The authors wish to thank the State Education Commission of China for supporting this research. REFERENCES Aunstrup, K. (1980). Proteinases. In Microbial Enzymes and Bioconversions (ed. A. H. Rose), pp. 50–114. Academic Press : London, New York, Toronto, Sydney and San Francisco. Burlini, N., Magnani, P., Villa, A., Macchi, F., Tortora, P. & Guerritore, A. (1992). A heat-stable serine proteinase from the extreme thermophilic archaebacterium Sulfolobus solfataricus. Biochimica et Biophysica Acta 1122, 238–292. (Accepted 19 April 1996)
Cooney, D. G. & Emerson, R. (1964). Thermophilic Fungi. An Account of their Biology, Activities, and Classification. W. H. Freeman : San Francisco and London. Connaris, H., Cowan, D. A. & Sharp, R. J. (1991). Heterogeneity of proteinases from the hyperthermophilic archaeobacterium Pyrococcus furiosus. Journal of General Microbiology 137, 1193–1199. Cowan, D. A., Smolenski, K. A., Daniel, R. M. & Morgan, H. W. (1987). An extremely thermostable extracellular proteinase from a strain of the archaebacterium Desulfurococcus growing at 88 °C. Biochemical Journal 247, 121–133. Domsch, K. H., Gams, W. & Anderson, T. H. (1980). Compendium of Soil Fungi. Academic Press : London. Farley, P. C. & Ikasari, L. (1992). Regulation of the secretion of Rhizopus oligosporus extracellular carboxyl proteinase. Journal of General Microbiology 138, 2539–2544. Haasum, I., Eriksen, S. H., Jensen, B. & Olsen, J. (1991). Growth and glucoamylase production by the thermophilic fungus Thermomyces lanuginosus in a synthetic medium. Applied Microbiology and Biotechnology 34, 656–660. Jensen, B., Wiebe, M. G., Robson, G. D., Trinci, A. J. P. & Olsen, J. (1993). Growth kinetics of the thermophilic fungus Thermomyces lanuginosus. Mycological Research 97, 665–669. Kalisz, H. M., Wood, D. A. & Moore, D. (1987). Production, regulation and release of extracellular proteinase activity in Basidiomycete fungi. Transactions of the British Mycological Society 88, 221–227. Kristjansson, J. K. (1989). Thermophilic organisms as sources of thermostable enzymes. Trends in Biotechnology 7, 349–353. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T . Nature 227, 680–685. % Lasure, L. L. (1980). Regulation of extracellular acid protease in Mucor miehei. Mycologia 72, 483–493. Lin, X.-L. & Tang, J. (1990). Purification, characterization and gene cloning of Thermopsin, a thermostable acid protease from Sulfolobus acidocaldarius. Journal of Biological Chemistry 265, 1490–1495. North, W. J. (1982). Comparative biochemistry of the proteinases of eucaryotic microorganisms. Microbiological Reviews 46, 308–340. Ong, P. S. & Gaucher, G. M. (1973). Protease production by thermophilic fungi. Canadian Journal of Microbiology 19, 129–133. Ong, P. S. & Gaucher, G. M. (1975). Production, purification and characterization of thermomycolase, the extracellular serine protease of the thermophilic fungus Malbranchea pulchella var. sulfurea. Canadian Journal of Microbiology 22, 165–176. Outtrup, H. & Boyce, C. O. L. (1990). Microbial proteinases and biotechnology. In Microbial Enzymes and Biotechnology, 2nd edn. (ed. W. M. Fogarty), pp. 227–254. Elsevier Science Publishers : London and New York. Rick, W. (1974). Chymotrypsin. In Methods of Enzymatic Analysis (ed. H. U. Bergmeyer), pp. 1045–1051. Academic Press : Berlin, New York, and London. Vogel, H. J. (1956). A convenient growth medium for Neurospora (Medium N). Microbial Genetics Bulletin 13, 42–44. Ward, O. P. (1983). Proteinases. In Microbial Enzymes in Biotechnology (ed. W. M. Fogarty), pp. 251–317. Applied Science Publishers : London.
18
Printed in Great Britain
Protease production by the thermophilic fungus Thermomyces lanuginosus
D U O - C H U A N L I, Y I - J U N Y A N G A N D C H O N G -Y A O S H E N Department of Plant Pathology, China Agricultural University, Beijing 100094, P.R. China
A total of 500 strains of the thermophilic fungus Thermomyces lanuginosus were screened for their ability to produce proteases. A selected strain, T. lanuginosus P , showed high enzymatic activity for proteases. The maximal yield of proteases was obtained with "$% 4 % casein, 4 % glucose and 4 % yeast extract in submerged culture. The protease system was shown to contain at least two protease isoenzymes by SDS–PAGE with gels containing gelatin as protease substrate. The effects of inhibitors imply that the two enzymes appear to be serine proteases. The crude proteases of T. lanuginosus P displayed unusual properties. The proteases were shown to "$% be one of the most thermostable proteases so far isolated in fungi. The proteases were 100 % stable at 50 °C. The half-life at 60 and 70° was approximately 160 min and 60 min, respectively. Temperature optimum of activity was 70°. The crude proteases were stable over a broad pH range (4–11). Optimum pH of activity was at 5±0 and 9±0.
Proteases are an important group of enzymes both physiologically and commercially. Microbial proteases dominate commercial applications (Ward, 1983 ; Outtrup & Boyce, 1990). A major requirement for commercial enzymes is thermal stability, because thermal denaturation is a common cause of enzyme inactivation (Kristjansson, 1989). In recent years, there has been increasing interest in proteases from thermophiles, which were expected to produce thermostable proteases. From known reports, most of the research has been on proteases from thermophilic bacteria of the genera Bacillus and Thermus, and reports from thermophilic fungi are limited (Ong & Gaucher, 1973, Aunstrup, 1980 ; Ward, 1983 ; Kristjansson, 1989 ; Outtrup & Boyce, 1990). The thermophilic Thermomyces lanuginosus Tsikl. (formerly Humicola lanuginosa ; Domsch, Gams & Anderson, 1980), has been shown to produce proteases (Ong & Gaucher, 1973), but detailed information is sparse. In this paper we report on the production, identification and partial characterization of the crude proteases by T. lanuginosus.
previously (Cooney & Emerson, 1964). Identification was conducted mainly according to the taxonomical methods of Cooney & Emerson (1964) and Domsch et al. (1980). Selection. All strains isolated were maintained on YpSs agar. A strain P with high protease activity was selected by "$% inoculating isolates onto yeast extract agar plates with 1 % skim milk. Growth conditions. T. lanuginosus was grown in shake cultures at 50 °C on a modified medium from Ong & Gaucher (1973) containing the following (g l−") : varying amounts of casein, glucose and yeast extract (Table 1) ; 1 g Table 1. Effect of concentration of casein, glucose, and yeast extract on protease production by T. lanuginosus. The crude proteases were from 12d-old submerged cultures of T. lanuginosus at 50° Protease activity (µ ml−")*
MATERIALS AND METHODS
Treatment medium
pH 5±0
pH 9±0
Materials. Casein, gelatin, iodoacetamide, acrylamide, bisacrylamide, bromophenol blue, Tris, glycine and tyrosine were from Sigma, PMSF, EDTA, Coomassie brilliant R , SDS, #&! TEMED, Triton X-100 and ammonium persulphate were from Serva, Yeast extract was from Difco, and soluble starch was from Merck. Other reagents were of analytic grade and were obtained from Beijing Chemical Reagent Corporation (Beijing, China).
4 % casein, 0±6 % glucose, 0±4 % yeast extract 4 % casein, 0±4 % glucose, 0±4 % yeast extract 4 % casein, 0±2 % glucose, 0±4 % yeast extract 4 % casein, 0±4 % yeast extract 4 % casein, 0±4 % glucose, 0±6 % yeast extract 4 % casein, 0±4 % glucose, 0±2 % yeast extract 4 % casein, 0±4 % glucose 4 % soluble starch, 0±4 % yeast extract 4 % glucose, 0±4 % yeast extract 2 % casein, 0±4 % glucose, 0±4 % yeast extract 6 % casein, 0±4 % glucose, 0±4 % yeast extract 0±4 % glucose, 0±4 % yeast extract
8±0³0±3 14±0³0±2 10±5³0±6 9±0³0±6 12±8³0±3 11±2³0±5 4±5³0±6 3±0³0±5 3±0³0±3 10±0³0±7 6±0³0±2 1±7³0±3
8±6³0±1 12±3³0±1 11±0³0±3 7±2³0±2 10±0³0±2 9±5³0±1 3±6³0±2 3±5³0±2 4±0³0±9 7±0³0±4 4±3³0±7 2±2³0±6
Isolation and identification. Strains of T. lanuginosus were isolated from compost, dung and soil on Emerson’s YpSs (Yeast Extract Soluble Starch) agar by a method described
* Values are the mean³... of two experiments with three replicates each.
D.-C. Li, Y.-J. Yang and C.-Y. Shen K HPO \3H O ; 0±5 g MgSO \7H O ; 0±1 ml of Vogel’s # % # % # trace element solution (Vogel, 1956). The pH was adjusted to 7±4 with 1 N NaOH. After different times, the mycelium was filtered off, then the culture filtrate was centrifuged at 10 000 g for 30 min at 4°, and the resultant supernatant was dialysed overnight against three changes of water. The dialysed sample of crude proteases was stored at 0°. Enzyme assays. The protease activity of the crude proteases was determined by using the modified Rick’s method (Rick, 1974). The enzymes (0±5 ml) were added to 2±5 ml of 0±4 % casein in 0±2 Tris}HCl (pH 9±0) or 0±2 CH COOH} $ CH COONa (pH 5±0) buffer and incubated at 50° for $ 60 min. Two ml 10 % TCA (Trichloroacetic acid) was then added, and after standing at room temperature for 30 min, the solution was filtered. To 1 ml of filtrate, 5 ml of water was added, the optical density (O.D.) at 280 nm was determined spectrophotometrically in UV-160A (Shimadzu). The blank used was prepared by adding 10 % TCA to the enzyme before addition of casein. One unit of protease activity was defined as the amount of enzyme producing 1 µg tyrosine min−" under assay conditions. Electrophoretic procedures. SDS–PAGE was used to establish the number of electrophoretically distinct proteases, to follow changes in protease profiles during growth, and to identify the protease isoenzymes of T. lanuginosus. Electrophoretic separation of proteases was performed in 4 % stacking}10 % separation gel. The separation gel composition was as follows : acrylamide (30 %) and bisacrylamide (1 %) in distilled water 3±3 ml ; 1±5 Tris}HCl (pH 8±8), containing 0±4 % SDS 2±25 ml ; gelatin (1 %) in distilled water 0±9 ml ; ammonium persulphate (10 %) in water 0±02 ml ; TEMED 0±01 ml ; water 2±45 ml. The stacking gel composition was as follows : acrylamide (30 %) and bisacrylamide (1 %) in distilled water 0±2 ml ; 0±5 Tris}HCl (pH 6±8), containing 0±4 % SDS 0±25 ml ; ammonium persulphate (10 %) in water 0±02 ml ; TEMED 0±01 ml ; water 1±55 ml. Samples of equal volumes in sample buffer (0±1 Tris}HCl, pH 6±8, containing 2 % (w}v) SDS, 10 % (v}v) glycerol and bromophenol blue as front marker) were electrophoresed at 10 mA constant current at 4° for 3 h, using the buffer system described by Laemmli (1970). After separation proteins were renatured by washing the gels in 50 m Tris}HCl (pH 9±0) or 50 m CH COOH}CH$ COONa (pH 5±0), containing 5 % (v}v) Triton X-100 twice $ for 15 min at 4°, followed by one 15 min wash at 4° in the same buffer without Triton X-100. The gels were then incubated in 50 m Tris}HCl (pH 9±0) or 50 m CH COOH} $ CH COONa (pH 5±0) for 3 h at 50° to allow degradation of $ the gelatin. Gels were stained in 0±1 % (w}v) Coomassie brilliant blue G250 reagent in 45 % (v}v) methanol and 10 % (v}v) acetic acid and destained in 30 % (v}v) methanol and 10 % (v}v) acetic acid. For further identification different inhibitors specific for serine-proteases (phenylmethyl sulphonyl fluoride, PMSF), cystein-proteases (iodoacetamide), and metallo-proteases (ethylenediaminetetraacetic acid, EDTA) were incubated with the enzymes. PMSF (0±5 m) was added to the sample and incubated for 25 min on ice prior to electrophoresis.
19 Iodoacetamide (0±5 m) and EDTA (30 m) were added to the gel washes and were also present during gel incubation. Protease partial characterization. The influence of pH on protease activity was determined by incubating the enzyme with casein for 60 min at 50° in the presence of buffers of a wide pH range (pH 2–12). The buffers used were : 0±2 HCl}KCl (pH 2–3) ; 0±2 CH COOH}CH COONa $ $ (pH 4–5) ; 0±2 NaH PO }Na HPO (pH 6–7) ; 0±2 Tris} # % # % NaOH (pH 8–12). Activity was estimated as a percentage of the maximum. Temperature optimum was determined by incubating the proteases with casein and buffer for 60 min at different temperatures. Thermal stability of proteases was examined in the range 50–80° at pH 5±0 and pH 9±0. The proteases were incubated in buffers without addition of casein, and samples were removed at fixed time intervals and allowed to cool on ice before the residual activities were determined under standard conditions. The buffers used were 0±2 CH COOH} $ CH COONa (pH 5±0) and 0±2 Tris}HCl (pH 9±0). Activity $ was measured as a percentage of the maximum. The protease inhibitors, PMSF, iodoacetamide and EDTA, were used to determine the effects on protease activity. PMSF was dissolved in 100 % methanol ; EDTA and iodoacetamide were dissolved in distilled water. The enzymes were preincubated with each inhibitor at either pH 5±0 or 9±0 for 1 h at room temperature. The samples were subsequently added to the buffer with casein for 60 min at 50°. Control with distilled water and methanol was treated in the same way as the test samples. The buffer used was 0±2 CH COOH} $ CH COONa (pH 5±0) and 0±2 Tris}HCl (pH 9±0). $ RESULTS Isolation and identification of T. lanuginosus About 500 strains of T. lanuginosus were isolated and identified. The strains isolated were characterized by colonies on YpSs media reaching a diameter of 9 cm in 5 d at 50°, white, later becoming grey, and eventually black, with a pink to vinaceous pigment diffusing into the agar. Aleurioconidia arising on lateral stalk cells, singly, at first hyaline, then becoming dark brown, globose, 5–9 µm diam., thick-walled, verruculose. Good growth occurred at temperatures between 40 and 55°, the minimum was about 35°, the optimum 50° and the maximum about 60°. Selection of a high-yield protease strain of T. lanuginosus Following growth for 72 h on yeast extract agar plates with 1 % skim milk, a clearing zone was observed around the colonies, indicating protein degradation. Among 500 strains, P strain produced the widest clearing zone. The strain was "$% selected for further studies. Influence of the carbon and nitrogen on extracellular protease production Four percent (w}v) casein, 0±4 % (w}v) glucose, and 0±4 %
Production of proteases by Thermomyces lanuginosus
20
14
12
12
10
10
8
8
6
6
4
4
2
2
0
0 14
0
2
4 6 8 10 Time since inoculation (d)
12
100 Protease activity (%)
14
pH Dry weight (g ml–1)
Protease activity (U ml–1)
16
Time course of extracellular protease production The time course of extracellular protease production from submerged cultures grown on 4 % casein, 0±4 % glucose and 0±4 % yeast extract of T. lanuginosus at 50° is shown in Fig. 1. It is clear from these experiments that protease formation was associated with mycelial growth. Large increases in protease activity paralleled rapid increases in mycelial biomass. Protease activity in the medium reached a maximum at 12 d. At the same time the maximum dry weight was 5±1 gl−". The pH dropped slowly to 5±8 at 4 d followed by an increase to a maximum at 12 d. SDS–PAGE profiles and isoenzymes of proteases produced at various growth times The samples from different growth time were separated by SDS–PAGE into two proteolytic major bands active at pH 5±0 and pH 9±0 (Fig. 2). The isoenzymes were designated protease I and protease II, according to their electrophoretic mobility, with protease I having the lower mobility. 10
40
0
(w}v) yeast extract gave the highest yield of proteases (Table 1).
12
60
20
Fig. 1. Time course of growth and protease production by T. lanuginosus. The crude proteases were from submerged cultures grown on 4 % casein, 0±4 % glucose, and 0±4 % yeast extract of T. lanuginosus at 50° at different growth stages. Activity was assayed as in experimental procedure. +, activity (at pH 5±0) ; *, activity (at pH 9±0) ; E, dry weight ; _, pH. Values are the mean³... of two experiments with three replicates each. Error bars show ...
14
80
8
pH 9.0
6
4
2
2
4
6
8
10
12
14
pH
Fig. 3. Effect of pH on protease activity of T. lanuginosus. The crude proteases were from 12-d-old submerged cultures of T. lanuginosus grown on 4 % casein, 0±4 % glucose and 0±4 % yeast extract at 50°. Activity was estimated at pH 2–12 and 50° for 1 h. Values are the mean³... of two experiments with three replicates each. Error bars show ...
Protease I appeared at day 4, and reached a maximum at 12 d (Fig. 2). Protease II presented at 6 d and reached a maximum at 12 d. Clearly, the profiles observed in the gel (Fig. 2) are consistent with the changes in activity shown in Fig. 1. Partial characterization of T. lanuginosus P134 proteases The sample from 12 day old submerged cultures grown on 4 % casein exhibited optimal activity from 4±0 to 11±0. Outside this range, activity was lost rapidly. Optimum pH of the sample was at 5±0 and 9±0 (Fig. 3). Activity increased rapidly from 40° to a maximum at 70°, with rapid loss of activity at temperatures above 70° (Fig. 4). At 50° the proteases were 100 % stable (Fig. 5). The halflife of the proteases at 60 and 70° was approximately 160 min and 60 min, respectively, and at 80° the activity was lost within 5 min. Thermostability at pH 9±0 was similar (data not shown). The proteolytic activity of the sample was completely inhibited at acid and alkaline pH values by PMSF, and not inhibited by iodoacetamide and EDTA (Table 2). This indicates the presence of serine protease(s) within the sample, with little or no cysteine protease(s) and metalloprotease(s). 14
12
10
8
6
4
2
pH 5.0
Fig. 2. Patterns of Thermomyces lanuginosus proteases after different periods of growth in submerged cultures (2, 4, 6, 8, 10, 12, 14 d). After electrophoretic separation the gel was incubated remaining substrate was stained, and destained. The assays were carried out at 50 m CH COOH}CH COONa (pH 5±0) and 50 m Tris}HCl (pH 9±0). $ $
D.-C. Li, Y.-J. Yang and C.-Y. Shen
21 Table 2. Effect of inhibitors on protease activity of T. lanuginosus. The crude proteases were from 12-d-old submerged cultures grown on 4 % casein, 0±4 % glucose and 0±4 % yeast extract of T. lanuginosus at 50°. The crude proteases were pre-incubated with each inhibitor at either pH 5±0 or pH 9±0 for 1 h at room temperature
Protease activity (%)
100 80
Inhibition (as % of control)*
60
Inhibitor
pH 5±0
pH 9±0
40
PMSF (0±5 m) Iodoacetamide (0±5 m) EDTA (30 m)
100 (100³1±32) 0 (0±21³0±08) 0 (0±14³0±02)
100 (100³1±78) 0 (0±39³0±04) 0 (0±36³0±03)
* Values are the mean³... of two experiments with three replicates each.
20
0
20
40
60 80 Temperature (C)
100
120
Fig. 4. Effect of temperature on protease activity of T. lanuginosus. The crude proteases were from 12-d-old submerged cultures of T. lanuginosus grown on 4 % casein, 0±4 % glucose, and 0±4 % yeast extract at 50°. Activity was estimated at pH 5±0 (+) and pH 9±0 (E) at each temperature for 1 h. Values are the mean³... of two experiments with three replicates each. Error bars show ...
120
Protease activity (%)
100 80 60 40 20
0
50
100 150 Time (min)
200
250
Fig. 5. Thermal stability of proteases from T. lanuginosus. The crude proteases were from 12-d-old submerged cultures of T. lanuginosus grown on 4 % casein, 0±4 % glucose and 0±4 % yeast extract at 50°. The crude proteases were pre-incubated in buffers without addition of casein, and samples were removed at fixed time intervals and allowed to cool on ice before the residual activities were determined at pH 5±0 at 50° for 1 h. +, 50° ; *, 60° ; _, 70° ; E, 80°. Values are the mean³... of two experiments with three replicates each. Error bars show ...
Inclusion of the inhibitors iodoacetamide and EDTA in the protease gel assay did not appear to alter the enzyme pattern of the crude proteases at pH 5±0 and pH 9±0 (Fig. 6). In contrast, PMSF reduced the intensities of two protease main bands (protease I and protease II) at pH 5±0 and pH 9±0, indicating that protease I and protease II are serine proteases. DISCUSSION Regulation of extracellular protease production has been studied in a number of fungi and normally involves either
induction or repression}depression or a combination of both (Lasure, 1980 ; North, 1982 ; Kalisz, Wood & Moore, 1987 ; Farley & Ikasari, 1992). In this study, extracellular protease production by T. lanuginosus was induced by protein such as casein and not induced by starch. Higher concentrations of casein yielded lower amounts of enzyme, presumably because of degradation of casein to amino acids by enzyme in the medium, and subsequent repression of enzyme production by amino acids. Glucose repressed extracellular protease production, though a small amount of glucose increased protease production. Similar observations have been made previously (Ong & Gaucher, 1973). Protease production was also stimulated by yeast extract, but higher concentration of yeast extract diminished protease production. This is likely because yeast extract increased conidum germination and mycelial growth of T. lanuginosus (Haasum et al., 1991 ; Jensen et al., 1993), which increased protease production ; on the other hand, amino acids in yeast extract repressed protease production, which decreased protease production. Proteases are a complex group of enzymes varying greatly in their physicochemical and catalytic properties. They are generally classified by pH optimum and catalytic mechanism. On the basis of pH optimum, proteases are classified into acid, neutral, and alkaline proteases. Acid, neutral and alkaline proteases have pH optima of 2±0–5±0, 7±0–8±0 and 7±0–11±0, respectively. On the basis of catalytic mechanism, proteases are classified into serine proteases, cysteine proteases, aspartic proteases and metalloproteases, determined indirectly through reactivity towards inhibitors of particular amino acid residues in the active site region (North, 1982). With respect to pH optimum, T. lanuginosus P proteases had a very broad pH "$% optimum. There was little or no difference in properties between at pH 5±0 and at pH 9±0. Therefore, T. lanuginosus P proteases cannot be classified into acid, neutral or alkaline "$% proteases. With respect to inhibitor sensitivity, T. lanuginosus P proteases were inhibited by the serine inhibitor PMSF, "$% but not by thiol reagents such as iodoacetamide. Therefore, the proteases are classified as serine proteases. Ong & Gaucher (1973) reported that 20 and 80 % inhibition of extracellular proteases of T. lanuginosus was caused by DFP and pCMB, respectively. This suggests the presence of serine proteases, but it is not definite whether there were cysteine proteases, since many serine proteases in fungi are also inhibited by thiol reagents (North, 1982). In our experiments, the assays showed 100 % inhibition
Production of proteases by Thermomyces lanuginosus 1
2
3
22 4
1
2
3
GHOST IMAGE
GHOST IMAGE
pH 9.0
pH 5.0
4
Fig. 6. Effect of different inhibitors on Thermomyces lanuginosus proteases. Inhibitors of cysteine proteases (Iodoacetamide, 0±5 m ; lane 2) and metalloproteases (EDTA, 30 m ; lane 3) were added to the gel washing and incubation buffers. Inhibitor of serine proteases (PMSF, 0±5 m ; lane 4) was added to the samples before SDS–PAGE. Untreated proteases served as control (lane 1). The assays were carried out at 50 m CH COOH}CH COONa (5±0) and 50 m Tris}HCl (9±0). $ $
(Table 2) and the gels showed only partial inhibition (Fig. 6). There are two possible reasons for this : (i) sensitivity of the assays is lower than that of the gels ; (ii) reaction time of the assays is shorter than that of the gels. There is possibly not 100 % inhibition in the assays, but because reaction time is short and product formed is very low, the difference in activity is not detected. The proteases from T. lanuginosus P described in this "$% paper displayed a high thermal stability as compared with those of other proteases in fungi (Aunstrup, 1980). The proteases were proven to be some of the most thermostable so far isolated in fungi, though not as much as those exhibited by bacteria such as Bacillus amyloliquefaciens (Aunstrup, 1980), Sulfolobus solfataricus (Burlini et al., 1992), Desulfurococcus mucosus (Cowan et al., 1987), Sulfolobus acidocaldarius (Lin & Tang, 1990), and Pyrococcus furiosus (Connaris, Cowan, & Sharp, 1992). The half-life of the proteases at 70° was 60 min in the absence of Ca#+. To our knowledge, among fungal proteases only the extracellular serine protease from the thermophilic fungus Malbranchea pulchella var. sulfurea displays a comparable thermostability. The half-life of the serine protease at 73° was 7±5 min in the absence of added Ca#+ and increased to 110 min upon addition of Ca#+ (Ong & Gaucher, 1975). The molecular basis of thermostability is still poorly understood. The hydrophobic bonds, ionic interactions and disulphide bridges in the enzyme molecule may be involved in thermostability (Kristjansson, 1989). From the characteristics of T. lanuginosus P proteases, it "$% would appear that the proteases may be of potential value in industry. Further studies on proteases of T. lanuginosus P "$% are necessary. Purification of the proteases is in progress in our laboratory. The authors wish to thank the State Education Commission of China for supporting this research. REFERENCES Aunstrup, K. (1980). Proteinases. In Microbial Enzymes and Bioconversions (ed. A. H. Rose), pp. 50–114. Academic Press : London, New York, Toronto, Sydney and San Francisco. Burlini, N., Magnani, P., Villa, A., Macchi, F., Tortora, P. & Guerritore, A. (1992). A heat-stable serine proteinase from the extreme thermophilic archaebacterium Sulfolobus solfataricus. Biochimica et Biophysica Acta 1122, 238–292. (Accepted 19 April 1996)
Cooney, D. G. & Emerson, R. (1964). Thermophilic Fungi. An Account of their Biology, Activities, and Classification. W. H. Freeman : San Francisco and London. Connaris, H., Cowan, D. A. & Sharp, R. J. (1991). Heterogeneity of proteinases from the hyperthermophilic archaeobacterium Pyrococcus furiosus. Journal of General Microbiology 137, 1193–1199. Cowan, D. A., Smolenski, K. A., Daniel, R. M. & Morgan, H. W. (1987). An extremely thermostable extracellular proteinase from a strain of the archaebacterium Desulfurococcus growing at 88 °C. Biochemical Journal 247, 121–133. Domsch, K. H., Gams, W. & Anderson, T. H. (1980). Compendium of Soil Fungi. Academic Press : London. Farley, P. C. & Ikasari, L. (1992). Regulation of the secretion of Rhizopus oligosporus extracellular carboxyl proteinase. Journal of General Microbiology 138, 2539–2544. Haasum, I., Eriksen, S. H., Jensen, B. & Olsen, J. (1991). Growth and glucoamylase production by the thermophilic fungus Thermomyces lanuginosus in a synthetic medium. Applied Microbiology and Biotechnology 34, 656–660. Jensen, B., Wiebe, M. G., Robson, G. D., Trinci, A. J. P. & Olsen, J. (1993). Growth kinetics of the thermophilic fungus Thermomyces lanuginosus. Mycological Research 97, 665–669. Kalisz, H. M., Wood, D. A. & Moore, D. (1987). Production, regulation and release of extracellular proteinase activity in Basidiomycete fungi. Transactions of the British Mycological Society 88, 221–227. Kristjansson, J. K. (1989). Thermophilic organisms as sources of thermostable enzymes. Trends in Biotechnology 7, 349–353. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T . Nature 227, 680–685. % Lasure, L. L. (1980). Regulation of extracellular acid protease in Mucor miehei. Mycologia 72, 483–493. Lin, X.-L. & Tang, J. (1990). Purification, characterization and gene cloning of Thermopsin, a thermostable acid protease from Sulfolobus acidocaldarius. Journal of Biological Chemistry 265, 1490–1495. North, W. J. (1982). Comparative biochemistry of the proteinases of eucaryotic microorganisms. Microbiological Reviews 46, 308–340. Ong, P. S. & Gaucher, G. M. (1973). Protease production by thermophilic fungi. Canadian Journal of Microbiology 19, 129–133. Ong, P. S. & Gaucher, G. M. (1975). Production, purification and characterization of thermomycolase, the extracellular serine protease of the thermophilic fungus Malbranchea pulchella var. sulfurea. Canadian Journal of Microbiology 22, 165–176. Outtrup, H. & Boyce, C. O. L. (1990). Microbial proteinases and biotechnology. In Microbial Enzymes and Biotechnology, 2nd edn. (ed. W. M. Fogarty), pp. 227–254. Elsevier Science Publishers : London and New York. Rick, W. (1974). Chymotrypsin. In Methods of Enzymatic Analysis (ed. H. U. Bergmeyer), pp. 1045–1051. Academic Press : Berlin, New York, and London. Vogel, H. J. (1956). A convenient growth medium for Neurospora (Medium N). Microbial Genetics Bulletin 13, 42–44. Ward, O. P. (1983). Proteinases. In Microbial Enzymes in Biotechnology (ed. W. M. Fogarty), pp. 251–317. Applied Science Publishers : London.