World Journal of Microbiology & Biotechnology 10, 563-567

A thermostable, alkaline-active, keratinolytic proteinase from Chrysosporium

keratinophilum

I.N.S. Dozie, C.N. Okeke* and N.C. Unaeze Thermostable alkaline proteinase was produced by a strain of Chrysosporium keratinophilum when cultured in lactose/mineral salt medium incorporating keratin solubilized with DMSO. The proteinase, partially purified by cold-acetone precipitation followed by gel-filtration on Sephadex G-75, was optimally active at pH 9 and stable from pH 7 to 10 with over 90% relative residual activity after incubation at 25°C for 24 h. The optimum temperature for enzyme activity was 90°C at which the activity half-life was 30 min. Enzyme activity was stimulated by Fe z + and inhibited by 1,10 o-phenanthroline. Gel-filtration indicated an M r of 69 kDa. Key words: Chrysosporium keratinophilum, keratinolytic proteinase.

Although the keratinolytic proteinases from keratinophilic fungi are well documented (Yu et al. 1968, 1971; Higuchi & Takiuchi I980; Takiuchi et al. 1982; Asahi et a]. 1985; Wawrzkiewicz et at. 1987) there are few reports on the production of thermostable proteinases by this group. Highly thermostable proteinases have mainly been reported from bacterial species (Manachini et al. 1988; Takami et al. 1989). Keratinolytic activity is brown in some species of Chrysosporium (Jain & Agrawal 1980) but there has been no attempt to establish a thermostable form of keratinolytic proteinase from this genus. The physico-chemical properties of a thermostable alkaline keratinolytic proteinase, from a strain of Chrysosporium keratinophilum isolated from a compost of poultry litter, are the subjects of the present study.

Materials and Methods Test Isolate Chrysosporium keratinophilum, isolated from poultry litter compost by the hair-baiting technique (Orr 1969), was used in the present study. Identification of the isolate was based on the standard morphological description by Chabasse (1988) and was confirmed by Prof. D. Chabasse of the Centre Hospitalier Regional et Universitaire D'Angers, France. The fungus is maintained in our culture collection with the accession number Myc.05. The authors are with the Department of Microbiology, Faculty of Biological Sciences, P.M.B.0O6, University of Nigeria, Nsukka, Enugu State, Nigeria. * Corresponding author,

Preparation of Keratin 5ubstrate and Fermentation Medium Scrapings from cow horn were ground to fine powder in a mechanical grinder, defatted for 30 rnin at 37°C with chloroform/ methanol (1:1, v/v), stored in water with soap (5%, v/v) for 12 h at 42°C, washed several times with distilled water and dried at room temperature (25 to 30°C) (Wawrzkiewicz et al. 1987). The resultant powder was used as the substrate. For the fermentation medium, keratin substrate, 10g was refluxed at 100°C for 4 h with 500 ml DMSO, cooled, centrifuged at 3000 x g for 10 rain and the solubilized protein in the supematant precipitated by adding 2 vol. cold acetone ( - I0oc). After standing at 4°C for 2 h, the mixture was centrifuged at 6000 x g and the precipitate washed in 4 voI. water and resuspended in water to give 0.64 mg keratin mt i as measured by the Lowry method. Fermentation medium contained (g 1 2 keratin suspension): lactose, 5; MgSO4.7H20, 0.5; FeSO4.7H20, 0.01; ZnSO4.7H20, 0.005; and peptone, 5. The medium was sterilized by Seitz-filtration and dispensed 50 ml into each of several 500ml Erlenmeyer flasks. Each flask was inoculated with a suspension of 2 x 108 conidia, prepared in phosphate-buffered saline from a 7-day culture grown on Sabouraud agar, and then incubated at 30°C in an orbital shaker set at 80 rev.min 1. In a control experiment, flasks containing Sabouraud broth were also similarly inoculated. Two flasks were removed periodically, the culture broth filtered through Whatman No.1 filter paper and the retained mycelial mass dried for 24 h at 80°C and weighed. The filtrate was centrifuged at 6000 x g at 4°C and the supematant used as the crude enzyme solution. Assays Protein content of the supematant was determined by the Lowry method. Keratinotytic proteinase activity was measured at 40°C by the method of Yu et al. (1968) but using pH 9. The assay mixture, containing 1.0 ml enzyme solution, 4.0 ml glycine/NaOH buffer

@ 1994 Rapid Communications of Oxford Ltd

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I.N.S. Dozie, C.N. Okeke and N.C. Unaeze 8O

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Figure 1. Mycelial growth ( 0 ) and keratinolytic proteinase activity ( 0 ) of Ch. keratinophi/um cultured in lactose/keratin/mineral salts medium.

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Figure 2. Sephadex G 75 chromatography of crude keratinolytic proteinase from Ch. keratinophilum, showing absorbance at 280 nm (@) and keratinolytic proteinase activity (©) in the fractions.

(pH 9.0) and 25.0 mg keratin substrate, was incubated at 40"C for 1 h. The reaction was terminated by adding 5.0 ml 0.6 M trichloroacetic acid and the mixture centrifuged at 3000 x g for 5 rain. The proteolytic products in the supernatant were determined from the absorbance at 280 nm against a control using an enzyme solution inactivated by boiling in a water bath for 30 min. An increase in absorbancy of 0.01 was taken to indicate I unit of enzyme activity m1-1. h.

Figure 3. Effect of pH on the activity (@) and stability ((3) of keratinolytic proteinase from Ch. keratinophilum. Buffers used were 0.02 M glycine/HCI (pH 3), 0.02 M sodium acetate (pH 4 to 6), 0.02 M KH=PO4/K=HPO, (pH 7 to 8), and 0.02 M glycine/NaOH (pH 9 to 10). Activities in the different buffer systems were determined as described in the text. For stability studies, equal volumes (1.0 ml) of the enzyme and the test buffer were mixed in a test tube, sealed with Parafiim and kept at 25°C for 24 h. The residual enzyme activity was determined at pH 9. 100% activity = 75 U ml 1.h.

were applied to a Sephadex G-75 (Pharmacia) column (3.3 x 93 cm) equilibrated with 0.2 M KHzPOJKzHPO4 buffer (pH 7.0) at 5"C. Fractions, 3.5 ml, were collected, monitored at 280 nm and assayed for keratinolytic proteinase activity. Active fractions from several gel filtration runs were pooled and dialysed against 100 vol. 0.02 ra KHzPOJKzHPO4 buffer (pH 7.0). The dialysate was split into 5-ml samples and frozen until needed for further tests. All tests were performed in triplicate and the data represent the means.

Partial Purification of Enzyme Protein was precipitated from the crude enzyme solution by adding two vol. chilled acetone ( - 10°C). The mixture was kept overnight at 4"C. The precipitated protein was solubilized in a minimal volume of 0.02 M KH2POJK2HPO4 buffer (pH 7.0), recovered by centrifugation at 10,000 x g for 15 rain and then resolubilized in the same buffer. About 6 ml of the crude enzyme

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Molecular Size Determination The M of the enzyme was determined by gel filtration on a Sephad~x G-75 column (3.3 × 93 cm), equilibrated with 0.02 M KH2POJK2HPO4 buffer (pH 7.0) and calibrated with L-lactic dehydrogenase (136 kDa), BSA (68 kDa), ovalbumin (43 kDa) and soy bean trypsin inhibitor (22 kDa).

Thermostable keratinasefrom Chrysosporium Table 1. Purification of keraUnolytic proteinase from Ch. keratinophilum. Purification procedure

Culture filtrate Acetone precipitate Gel filtration (Sephadex G 75)

Total enzyme activity (U)

Total protein (mg)

Sp. act.

Yield

[U (mg protein)- I]

(%)

Purification (fold)

8700 3850 1140

525 72 6

1.7 5.3 19

100 44.3 13

1 3 11

100 Table 2. Effect of some chemicals (at 1 mM) on the activity of the enzyme.* Chemical

90

Residual activity (% of control value)

None (control) EDTA 1,10 o-Phenanthroline Urea Co 2+ Hg 2+ Ba 2+ Ca =+ Mg =+ Fe =+ Zn =+

1001" 124 63 92 77 97 78 190 85 630 83

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40 * EDTA, 1,10 o-phenanthroline, urea and chlorides of the divalent metals were incorporated in the assay mixture and preincubated at room temperature (25 to 30°C) for 30 min before keratin substrate was added. 1" 100% = 75 U ml 1.h.

i

i

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600-

Temperature ( C )

m

70

i

i

80

90

100

Figure 4. Effect of temperature on the activity of keratinolytic proteinase from Ch. keratinophilum. Enzyme activity was assayed in the temperature range 40 to 100°C. 100% activity = 75 U ml l.h.

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Table 3. Activity of keratlnolytic protelnese from Ch. keratinophilum on keratin from different sources. 80 Keratin

Activity (U m1-1. h)

Chicken feather Guinea pig hair Cow hair Human scalp hair

57 59 71 60

60

.~ .o j 20

Activity of Keratinolytic Profeinase on Different Proteins

The ability of the enzyme to hydrolyse various native keratin sources and other proteins (casein, gelatin and BSA) was tested. Clean chicken feathers, human scalp hair, guinea pig hair and cow hair were cut into 1-K-2mm pieces and defatted as described previously (Wawrzkiewicz et al. I987). Activity was assayed as described above.

Results Keratinolytic proteinase activity was detected in the lactose/keratin/mineral salt culture filtrate after 4 days' growth of Ch. keratinophilum. The activity was maximum (80 U ml 1.hl) after 10 days (Figure 1). The pH of the culture rose gradually from an initial value of 8.0 to 10.4

O

10 20 Time (mini

30

40

50

60

Figure 5. Effect of temperature on the stability of keratinolytic proteinase from Ch. keratinophilum at 40°C ( © ) and 90°C (@). 1.0 ml of the enzyme solution was mixed with 4.0 ml of 0.02 M KH=PO4/K=HPO4 buffer, the tube sealed with Parafilm and incubated at either 40 or 90°C. Every 10 min, the tubes w e r e cooled rapidly in ice-cold water and the residual enzyme activity was determined as described in the text. Residual activity is expressed as a percentage of the control enzyme activity assayed at zero time. 100% activity = 75 U ml Lh.

after 8 days and thereafter decreased to 9.5. No enzyme activity was detected in culture filtrates of the fungus grown in Sabouraud dextrose broth. Gel-filtration of the crude enzyme (Figure 2) showed a single protein peak

World Journalof Microbiology & Biotechnology, Vol I0, I994

565

I.N.S. Dozie, C.N. Okeke and N.C. Unaeze corresponding with a single peak of keratinolytic proteinase activity. The enzyme recovery (Table 1) was 13% with 11fold purification. The enzyme was optimally active at pH 9.0 (Figure 3) and highly stable ( > 90% residual activity) from pH 7 to 10 at 25°C for 24 h. The optimum temperature for enzyme activity was 90°C (Figure 4). The proteinase had 100% activity retention after incubation at the assay temperature, 40°C, for 1 h whereas the activity half-life at 90°C was 30 min (Figure 5). Although Ca z + and Fe z + stimulated activity Ba2+, Co 2+, Mg 2+ and Zn z + were inhibitory to varying degrees and Hg 2+ had no effect (Table 2). The keratinolytic proteinase was inhibited by 1,10 0-phenanthroline by up to 42%; urea was slightly inhibitory and EDTA was slightly stimulatory. The Mr of the enzyme was estimated from gel-filtration on Sephadex G-75 to be 69 kDa. The enzyme only hydrolysed the keratin proteins (Table 3); no activity was recorded when casein, BSA or gelatin were used as substrates.

Discussion Chrysosporium keratinophilum produces a thermostable, alkaline-active, keratinolytic proteinase when grown in medium containing keratin as an exogenous inducer. When the fungus was grown in Sabouraud dextrose broth which lacked keratin, there was no activity. Similar inducibility of the keratinolytic proteinase of Candida albicans has been reported (Hattori et al. 1984). Exocellular and cell-bound keratinases have been purified from Trichophyton mentagrophytes (Yu et aL 1968, 1971). The keratinolytic proteinase had maximum activity at pH 9.0. It therefore differs from T. mentagraphytes (pH 7.0) (Yu et aL 1969), Microsporum gypseum (pH 7.8) (Higuchi & Takiuchi 1980), T. rubrum (pH 8.0) (Asahi et al. 1985) and T. gallinae (pH 8.0) (Wawrzkiewicz et al. 1987). The keratinolyric proteinase of the yeast, Ca. albicans, had maximum activity at pH 4.0 (Hattori et al. 1984). The enzyme was maximally active at 90°C. This compares with temperature optima of 40°C for the keratinolytic proteinase of M. gypseum (Higuchi & Takiuchi 1980), 43 to 54°C for that of T. mentagrophytes (Yu et aL 1969) and 45°C for that of T. granulosum (William et aL 1978). The thermostability studies indicated that the activity half-life of the enzyme at 90°C was 30 min. The enzyme would therefore be potentially useful for biotechnological applications, since a large number of such processes are carried out at elevated temperatures. The addition of Fe 2+ to the assay medium potentiated the activity by more than 5-fold. As expected, therefore, the iron chelator, 1.10 o-phenanthroline, was inhibitory. The Ch. keratinophilum keratinolytic proteinase may be a metalloproteinase with a requirement for iron.

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More tests on the pure enzyme are needed to confirm this. The Ca. albicans keratinase has been classified as a carboxyproteinase (Hattori et al. 1984). The molecular size of the Ch. keratinophilum enzyme, estimated at 69 kDa, lies within the range of those reported for fungal keratinolytic proteinases, i.e. 20.3 to 440 kDa (Yu et al. 1969, 1971). The enzyme was specific for keratin substrates; non-keratin proteins were not degraded. Alkaline proteolytic keratinases are of importance in leather tanning industries where they are used to remove hair from hides (Chaplin & Bucke 1990) in preference to traditional methods involving sodium sulphide. The enzymic bioconversion of vast quantities of keratin produced from poultry production to useful products such as protein fodder (Dalev 1990) is yet to be commercially exploited. This would also be an ecologically welcome enterprise.

References Asahi, M., Lindquist, R., Apodaca, G., Epstein, L.W. & McKerrows, J.H. 1985 Purification and characterisation of major extracellular proteinases from Trichophyton rubrum. BiochemicalJournal 232, 139-144. Chabasse, D. 1988 Taxonomic study of keratinophilic fungi isolated from soil and some mammals in France. Mycopathologia 101, 133-140. Chaplin, M.F. & Bucke, C. 1990 Enzyme Technology. Cambridge: Cambridge University Press. Dalev, P. 1990 An enzyme-alkaline hydrolysis of feather keratin for obtaining a protein concentrate for fodder. Biotechnology Letters 12, 71-72. Hattori, M., Yoshiura, K., Negi, M. & Ogawa, H. 1984 Keratino[ytic proteinase produced by Candida albicans. Sabouraudia 22, 175-183. Higuchi, M. & Takiuchi, I. 1980 Inhibitor of the purified extracellular keratinase of a Microsporum gypseum strain. JapaneseJournal of Medical Mycology 21, 101-108. Jain, P.C. & Agrawal, S.C. 1980 A note on the keratin decomposing capability of some fungi. Transactions of the Mycological Society of Japan 21, 513-517. Manachini, P.L., Fortina, M.G. & Parini, C. 1989 Thermostable alkaline protease produced by Bacillus thermoruber - - a new species of Bacillus. Applied Microbiology 28, 409-413. Orr, F.F. 1969 Keratinophilic fungi isolated from soils by a modified hair bait technique. Sabouraudia 7, 129-134. Takami, H., Akiba, T. & Horikoshi, K. 1989 Production of extremely thermostable alkaline protease from Bacillus sp. no. AH-101. Applied Microbiology and BiotechnoIogy 30, 120124. Takiuchi, I., Higuchi, D., Sei, Y. & Koga, M. 1982 Isolation of an extracellular proteinase (keratinase) from Microsporum canis. Sabouraudia 20, 281-288. Wawrzkiewicz, K., Labarzewski, J. & Wolski, T. 1987 Intracellular keratinase of Trichophytongallinae. Journal of Medical and Veterinary Mycology 25, 261-268. William, C.D., Tonic, P., Stratman, C., Leeman, U. & Harmon, S. 1978 Isolation and properties of extracellular protease of Trichopyton granulosum. Biochimica et Biophysica Acta 167, 597599. Yu, R.J., Harmon, S.R. & Blank, F. 1968 Isolation and purification

Thermostable keratinase from Chrysosporium of an extracellular of keratinase of Trichophyton mentagrophytes. Journal of Bacteriology 96, 1435-1436. Yu, R.J, Harmon, S.R. & Blank, F. 1969 Hair digestion by a keratinase of Trichophyton mentagrophytes. Journal of Investigative Dermatology 53, 166--171. Yu, R.J., Harmon, S.R., Grappel, S.F. & Blank, F. 1971 Two cell-

bound keratinase of Trichophyton mentagrophytes. Journal of Investigative Dermatology 56, 27-32.

(Received in revised form 12 M a y I994; accepted 16 M a y 1994)

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