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Biosynthesis and Activity of the Extracellular Proteases of Thermoascus aurantiacus Miehe from Poultry Feeds
Biosynthesis and Activity of the Extracellular Proteases of Thermoascus aurantiacus Miehe from Poultry Feeds VINCENT W. OGUNDERO Department of Botany, University of Ibadan, Ibadan, Nigeria (Received for publication September 13, 1982) ABSTRACT The production and activity of extracellular proteases of the zoopathogenic Thermoascus aurantiacus Miehe obtained from poultry feeds in Nigeria were studied. These enzymes were detected in culture filtrates of T. aurantiacus within 48 hr of incubation on a livestock feed medium at 45 C. Peak production occurred within the 10-day incubation period but after autolysis. With growth of the organism, increasing amounts of proteases were liberated into the medium. Optimal protease activity was obtained at pH 6.0 and 45 C. Because of the zoopathogenic nature of this fungus and the potential health risks posed to farm animals and man, careful handling of livestock feeds during and after processing is suggested. (Key words: fungus, Thermoascus aurantiacus miehe, protease, feeds) 1983 Poultry Science 62:763-766 INTRODUCTION
Microorganisms, including fungi, are responsible for the spoilage of agricultural products during storage (Mulinge and Apinis, 1969; Pounds and Lucas, 1972). This is particularly pronounced in the humid tropics where high rainfall and environmental temperatures provide excellent conditions for microbial colonization of such microorganisms. In an earlier survey of thermophilic and thermotolerant mycoflora of poultry droppings in Nigeria (Ogundero, 1979), several species were consistently obtained, including the zoopathogenic Thermoascus aurantiacus Miehe. A subsequent study (Ogundero, 1980) showed this fungus was not a contaminant of the droppings as previously thought but rather was present originally in the feeds fed to the birds. Studies were later carried out on the cultural and nutritional physiology of the fungus (Ogundero, 1981). This communication is on the production and activity of the extracellular acid proteases, of T. aurantiacus.
MATERIALS AND METHODS
The method of isolation and identification of T. aurantiacus has been previously described (Ogundero, 1979; 1980). Growth-temperature relations on livestock feed infusion broth were measured by growth at each of five temperatures (Fig. 1) for 5 days after which the mycelia produced were suction-filtered, oven-dried, and weighed. The livestock feed infusion broth was prepared by boiling 100 g feeds with 1000 ml
distilled water, cooling, and squeezing out the liquid content from muslin cloth. Broth was supplemented with 2.5 g yeast extract, made up to 1 liter with distilled water, and dispensed into 250-ml conical flasks (30 ml per flask). All flasks were then autoclaved at 121 C for 15 min. The pH after autoclaving was 7.1. The inoculum was obtained by growing the fungus on agar plates of the feed broth for 3 days after which young mycelia-agar discs (5 mm diameter) were cut out with a flamed cork-borer. Each flask was then inoculated with one disc before incubation. The results are means of three readings. Extracellular Protease Production and Activity. A livestock feed medium was used for this study. This was obtained by suspending 15 g of the feed in a liter of distilled water, casein (4.0 g) was added, and the mixture thoroughly shaken before 30-ml portions were dispensed into several 250-ml conical flasks. All flasks were then sterilized by autoclaving at 121 C for 15 min. Inclusion of casein in a growth medium for protease synthesis is known to enhance extracellular protease production by microfungi (Lasure, 1980). Each flask was inoculated with the test fungus and incubated as still cultures at 45 C. Uninoculated flasks served as control. At 2-day intervals, the protease activity of the culture filtrate was determined. One milliliter of culture filtrate was added to an assay mixture containing casein (4 ml of 1% wt/vol) in .05 M phosphate buffer (pH 6.0) and incubated for 1 hr at 45 C (McDonald and Chen, 1965). At the end of the incubation period, an
763
764
OGUNDERO
equal volume of 5% wt/vol) trichloroacetic acid (TCA) was added to precipitate the excess proteins. These were filtered throught two layers of glass fibre papers and the absorbance of the filtrates determined with a Beckman 25 Dual Beam spectrophotometer at 280 nm. A standard curve of increasing concentrations of tyrosine allowed conversion of absorbancy units to micromoles of tyrosine solubilized per milliliter. Filtrates from the control flasks, were similarly treated and used to set absorbance at zero. Changes in the pH of the growth medium were also followed, whereas protein contents of the filtrates were determined using the Folinphenol reagent method of Lowry et al. (1951). In a separate set-up, the fungus was grown on the livestock-feed broth earlier described at 45 C and the pH and mycelia dry weights measured with growth. Also, the effects of various natural products on protease synthesis by T. aurantiacus were determined by growth on corn-meal chaff, cowpea chaff, and groundnut meal chaff media at 45 C for 5 days- after which the protease activities of the culture filtrates were determined. The fungus was grown for 5 days on a chemically defined medium (D-glucose, 5.0 g; MgS0 4 ' 7 H 2 0 , .75 g; nitrogen source, 3.5 g; K H 2 P 0 4 , 1.75 g; distilled water, 1000 ml, pH 7.2) in which the nitrogen source was NaN0 3 and this served as the control. All salts are 'AR' grade (BDH chemicals). Filtrates from 5-day-old cultures growing on the feed medium at 45 C were used to assay the effects of pH on protease activity, the assay medium having been buffered to different pH (4.2 - 7.2) with citrate-phosphate buffers (Hale, 1958). The effects of temperature of incubation of the assay mixture on protease activity were also determined. Results of these studies are means of three readings.
25 35 TEMPERATURE
FIG. 1. Thermoascus aurantiacus. (A) Growthtemperature relations o o on livestock feed infusion broth after 5 days. (B) Mycelia dry weight (mg) • • with period of growth at 45 C. * * pH of growth medium.
near 50 C but with minima well below 20 C were termed thermotolerant. The ability of this fungus to make good growth at 37 C and above probably explains the relative ease with which the propagules grow in the lungs of farm animals and man (Cooney and Emerson, 1964) with similar body temperatures. Protease Production and Activity. Extracellular proteases were detected in the culture filtrates of T. aurantiacus within 48 hr of incubation at 45 C (Fig. 2) with peak production occurring on day 8 after autolysis had set in (Fig. IB). Similar results were obtained by Barnett and Fergus (1971) for the exo-enzymes of some thermophilic and
RESULTS AND DISCUSSION
Growth-Temperature Relations. T. aurantiacus had optimal mycelia growth at 45 C and on the 6th day of incubation (Fig. 1). Growth was much higher at 50 C than at 35 C. No growth occurred at 20 C and below. These results confirm the thermophilic nature of this fungus. According''to the working definition of Cooney and Emerson (1964), fungi with maximum growth-temperatures at or above 50 C and minima at or above 20 C were described as thermophilic, whereas those with maxima
0.
FIG. 2. Thermoascus aurantiacus. Protease activity • • of culture filtrate with period of incubation at 45 C. o o protein content of culture filtrate. * * pH.
THERMOASCUS AURANTIACUS
AND POULTRY FEED
765
FIG. 4. Thermoascus aurantiacus. Effects of pH • • and o o temperature of incubation of assay mixture on protease activity.
Cm
Cp Gn MEDIA
Lv
Ct
FIG. 3. Thermoascus aurantiacus. Effects of natural products (+ .4% casein a , —.4% casein D) on protease synthesis. Cultures were incubated at 45 C for 5 days. Cm, Corn meal chaff medium, Cp, cowpea chaff medium, Gn, groundnut meal chaff medium, Lv, livestock chaff medium, Ct, control.
mesophilic species of Humicola. With period of i n c u b a t i o n , proteins were excreted into t h e g r o w t h m e d i u m . T h e p H of t h e m e d i u m d r o p e d initially, followed b y a gradual rise w i t h t i m e . This is p r o b a b l y d u e t o an early rapid liberation of organic acids b y proteolysis b y t h e growing organism a t a rate faster t h a n t h e s u b s e q u e n t utilization. When t h e fungus was g r o w n on various natural p r o d u c t s t o see t h e effects t h e y e x e r t e d on protease synthesis (Fig. 3), best results were recorded o n t h e modified livestock feed m e d i u m . This confirms t h e earlier r e p o r t of Lasure ( 1 9 8 0 ) t h a t t h e presence of casein in culture m e d i u m e n h a n c e s protease synthesis. Similar results were o b t a i n e d o n t h e o t h e r natural p r o d u c t s (Fig. 3). Detection of p r o teases in t h e control flasks shows t h a t t h e presence of proteins in t h e g r o w t h m e d i u m is n o t needed t o induce t h e p r o d u c t i o n of these e n z y m e s . A d e t e r m i n a t i o n of t h e effects of t e m p e r a t u r e of incubation of assay m i x t u r e s and its p H o n protease activity (Fig. 4) shows t h a t p H 6.0 and a t e m p e r a t u r e of 4 5 C is o p t i m a l for t h e activities of t h e proteases of this fungus.
Thermoascus aurantiacus has been r e p o r t e d t o utilize native cellulose and o t h e r forms of carbon and nitrogen for g r o w t h and reprod u c t i o n (Tansey, 1 9 7 1 ) . In addition t o concentrates, these p r o d u c t s form t h e major part of livestock feeds. This organism can, therefore, be said t o be well suited t o t h e feed h a b i t a t from which it was o b t a i n e d . Because of t h e z o o p a t h o g e n i c n a t u r e of this m i c r o b e , p r o p e r handling of livestock feeds, particulary in t h e tropics, becomes necessary. REFERENCES Bamett, E. A., and C. L. Fergus, 1971. The relation of extracellular amylase, mycelium and time in some thermophilic and mesophilic Humicola species. Mycopathol. Mycol. Appl. 44:131—141. Cooney, D. G., and R. Emerson, 1964. Thermophilic Fungi. W. H. Freeman and Co., San Francisco, CA. Hale, L. J., 1958. Pages 8 0 - 8 9 in Biological Laboratory Data. Methuen and Co., Ltd., London. Lasure, L. L., 1980. Regulation of extracellular acid protease in Mucor miebei. Mycologia 72:483 — 493. Lowry, O. H., N. J. Rosebrough., A. L. Farr., R. J. Randall, 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 1 9 3 : 2 6 5 275. McDonald,. C. E., and L. L. Chen, 1965. The Lowry modification of the folin reagent for determination of proteinase activity. Ann. Biol. Chem. 10:175-177. Mulinge, S. K., and A. E. Apinis, 1969. Occurrence of thermophilous fungi from stored moist barley grains. Trans. Br. Mycol. Soc. 53:361-370. Ogundero, V. W., 1979. Thermophilic and thermo-
766
OGUNDERO
tolerant fungi in poultry droppings in Nigeria. J. Gen. Microbiol. 115:253-254. Ogundero, V. W., 1980. Fungal flora of poultry feeds. Mycologia 72:200-202. Ogundero, V. W., 1981. Cultural and nutritional studies of zoopathogenic fungi associated with livestock feeds in Nigeria Z. Allg. Mikrobiol.
21:255-259. Pounds, J. R., and G. B. Lucas, 1972. Thermophilic fungi of tobacco. Tech. Bull. No. 211, North Carolina Agric. Exp. Sta., Raleigh, NC. Tansey, M. R., 1971. Agar diffusion assay of cellulolytic ability of thermophilic fungi. Arch. Microbiol. 7 7 : 1 - 1 1 .
Microorganisms, including fungi, are responsible for the spoilage of agricultural products during storage (Mulinge and Apinis, 1969; Pounds and Lucas, 1972). This is particularly pronounced in the humid tropics where high rainfall and environmental temperatures provide excellent conditions for microbial colonization of such microorganisms. In an earlier survey of thermophilic and thermotolerant mycoflora of poultry droppings in Nigeria (Ogundero, 1979), several species were consistently obtained, including the zoopathogenic Thermoascus aurantiacus Miehe. A subsequent study (Ogundero, 1980) showed this fungus was not a contaminant of the droppings as previously thought but rather was present originally in the feeds fed to the birds. Studies were later carried out on the cultural and nutritional physiology of the fungus (Ogundero, 1981). This communication is on the production and activity of the extracellular acid proteases, of T. aurantiacus.
MATERIALS AND METHODS
The method of isolation and identification of T. aurantiacus has been previously described (Ogundero, 1979; 1980). Growth-temperature relations on livestock feed infusion broth were measured by growth at each of five temperatures (Fig. 1) for 5 days after which the mycelia produced were suction-filtered, oven-dried, and weighed. The livestock feed infusion broth was prepared by boiling 100 g feeds with 1000 ml
distilled water, cooling, and squeezing out the liquid content from muslin cloth. Broth was supplemented with 2.5 g yeast extract, made up to 1 liter with distilled water, and dispensed into 250-ml conical flasks (30 ml per flask). All flasks were then autoclaved at 121 C for 15 min. The pH after autoclaving was 7.1. The inoculum was obtained by growing the fungus on agar plates of the feed broth for 3 days after which young mycelia-agar discs (5 mm diameter) were cut out with a flamed cork-borer. Each flask was then inoculated with one disc before incubation. The results are means of three readings. Extracellular Protease Production and Activity. A livestock feed medium was used for this study. This was obtained by suspending 15 g of the feed in a liter of distilled water, casein (4.0 g) was added, and the mixture thoroughly shaken before 30-ml portions were dispensed into several 250-ml conical flasks. All flasks were then sterilized by autoclaving at 121 C for 15 min. Inclusion of casein in a growth medium for protease synthesis is known to enhance extracellular protease production by microfungi (Lasure, 1980). Each flask was inoculated with the test fungus and incubated as still cultures at 45 C. Uninoculated flasks served as control. At 2-day intervals, the protease activity of the culture filtrate was determined. One milliliter of culture filtrate was added to an assay mixture containing casein (4 ml of 1% wt/vol) in .05 M phosphate buffer (pH 6.0) and incubated for 1 hr at 45 C (McDonald and Chen, 1965). At the end of the incubation period, an
763
764
OGUNDERO
equal volume of 5% wt/vol) trichloroacetic acid (TCA) was added to precipitate the excess proteins. These were filtered throught two layers of glass fibre papers and the absorbance of the filtrates determined with a Beckman 25 Dual Beam spectrophotometer at 280 nm. A standard curve of increasing concentrations of tyrosine allowed conversion of absorbancy units to micromoles of tyrosine solubilized per milliliter. Filtrates from the control flasks, were similarly treated and used to set absorbance at zero. Changes in the pH of the growth medium were also followed, whereas protein contents of the filtrates were determined using the Folinphenol reagent method of Lowry et al. (1951). In a separate set-up, the fungus was grown on the livestock-feed broth earlier described at 45 C and the pH and mycelia dry weights measured with growth. Also, the effects of various natural products on protease synthesis by T. aurantiacus were determined by growth on corn-meal chaff, cowpea chaff, and groundnut meal chaff media at 45 C for 5 days- after which the protease activities of the culture filtrates were determined. The fungus was grown for 5 days on a chemically defined medium (D-glucose, 5.0 g; MgS0 4 ' 7 H 2 0 , .75 g; nitrogen source, 3.5 g; K H 2 P 0 4 , 1.75 g; distilled water, 1000 ml, pH 7.2) in which the nitrogen source was NaN0 3 and this served as the control. All salts are 'AR' grade (BDH chemicals). Filtrates from 5-day-old cultures growing on the feed medium at 45 C were used to assay the effects of pH on protease activity, the assay medium having been buffered to different pH (4.2 - 7.2) with citrate-phosphate buffers (Hale, 1958). The effects of temperature of incubation of the assay mixture on protease activity were also determined. Results of these studies are means of three readings.
25 35 TEMPERATURE
FIG. 1. Thermoascus aurantiacus. (A) Growthtemperature relations o o on livestock feed infusion broth after 5 days. (B) Mycelia dry weight (mg) • • with period of growth at 45 C. * * pH of growth medium.
near 50 C but with minima well below 20 C were termed thermotolerant. The ability of this fungus to make good growth at 37 C and above probably explains the relative ease with which the propagules grow in the lungs of farm animals and man (Cooney and Emerson, 1964) with similar body temperatures. Protease Production and Activity. Extracellular proteases were detected in the culture filtrates of T. aurantiacus within 48 hr of incubation at 45 C (Fig. 2) with peak production occurring on day 8 after autolysis had set in (Fig. IB). Similar results were obtained by Barnett and Fergus (1971) for the exo-enzymes of some thermophilic and
RESULTS AND DISCUSSION
Growth-Temperature Relations. T. aurantiacus had optimal mycelia growth at 45 C and on the 6th day of incubation (Fig. 1). Growth was much higher at 50 C than at 35 C. No growth occurred at 20 C and below. These results confirm the thermophilic nature of this fungus. According''to the working definition of Cooney and Emerson (1964), fungi with maximum growth-temperatures at or above 50 C and minima at or above 20 C were described as thermophilic, whereas those with maxima
0.
FIG. 2. Thermoascus aurantiacus. Protease activity • • of culture filtrate with period of incubation at 45 C. o o protein content of culture filtrate. * * pH.
THERMOASCUS AURANTIACUS
AND POULTRY FEED
765
FIG. 4. Thermoascus aurantiacus. Effects of pH • • and o o temperature of incubation of assay mixture on protease activity.
Cm
Cp Gn MEDIA
Lv
Ct
FIG. 3. Thermoascus aurantiacus. Effects of natural products (+ .4% casein a , —.4% casein D) on protease synthesis. Cultures were incubated at 45 C for 5 days. Cm, Corn meal chaff medium, Cp, cowpea chaff medium, Gn, groundnut meal chaff medium, Lv, livestock chaff medium, Ct, control.
mesophilic species of Humicola. With period of i n c u b a t i o n , proteins were excreted into t h e g r o w t h m e d i u m . T h e p H of t h e m e d i u m d r o p e d initially, followed b y a gradual rise w i t h t i m e . This is p r o b a b l y d u e t o an early rapid liberation of organic acids b y proteolysis b y t h e growing organism a t a rate faster t h a n t h e s u b s e q u e n t utilization. When t h e fungus was g r o w n on various natural p r o d u c t s t o see t h e effects t h e y e x e r t e d on protease synthesis (Fig. 3), best results were recorded o n t h e modified livestock feed m e d i u m . This confirms t h e earlier r e p o r t of Lasure ( 1 9 8 0 ) t h a t t h e presence of casein in culture m e d i u m e n h a n c e s protease synthesis. Similar results were o b t a i n e d o n t h e o t h e r natural p r o d u c t s (Fig. 3). Detection of p r o teases in t h e control flasks shows t h a t t h e presence of proteins in t h e g r o w t h m e d i u m is n o t needed t o induce t h e p r o d u c t i o n of these e n z y m e s . A d e t e r m i n a t i o n of t h e effects of t e m p e r a t u r e of incubation of assay m i x t u r e s and its p H o n protease activity (Fig. 4) shows t h a t p H 6.0 and a t e m p e r a t u r e of 4 5 C is o p t i m a l for t h e activities of t h e proteases of this fungus.
Thermoascus aurantiacus has been r e p o r t e d t o utilize native cellulose and o t h e r forms of carbon and nitrogen for g r o w t h and reprod u c t i o n (Tansey, 1 9 7 1 ) . In addition t o concentrates, these p r o d u c t s form t h e major part of livestock feeds. This organism can, therefore, be said t o be well suited t o t h e feed h a b i t a t from which it was o b t a i n e d . Because of t h e z o o p a t h o g e n i c n a t u r e of this m i c r o b e , p r o p e r handling of livestock feeds, particulary in t h e tropics, becomes necessary. REFERENCES Bamett, E. A., and C. L. Fergus, 1971. The relation of extracellular amylase, mycelium and time in some thermophilic and mesophilic Humicola species. Mycopathol. Mycol. Appl. 44:131—141. Cooney, D. G., and R. Emerson, 1964. Thermophilic Fungi. W. H. Freeman and Co., San Francisco, CA. Hale, L. J., 1958. Pages 8 0 - 8 9 in Biological Laboratory Data. Methuen and Co., Ltd., London. Lasure, L. L., 1980. Regulation of extracellular acid protease in Mucor miebei. Mycologia 72:483 — 493. Lowry, O. H., N. J. Rosebrough., A. L. Farr., R. J. Randall, 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 1 9 3 : 2 6 5 275. McDonald,. C. E., and L. L. Chen, 1965. The Lowry modification of the folin reagent for determination of proteinase activity. Ann. Biol. Chem. 10:175-177. Mulinge, S. K., and A. E. Apinis, 1969. Occurrence of thermophilous fungi from stored moist barley grains. Trans. Br. Mycol. Soc. 53:361-370. Ogundero, V. W., 1979. Thermophilic and thermo-
766
OGUNDERO
tolerant fungi in poultry droppings in Nigeria. J. Gen. Microbiol. 115:253-254. Ogundero, V. W., 1980. Fungal flora of poultry feeds. Mycologia 72:200-202. Ogundero, V. W., 1981. Cultural and nutritional studies of zoopathogenic fungi associated with livestock feeds in Nigeria Z. Allg. Mikrobiol.
21:255-259. Pounds, J. R., and G. B. Lucas, 1972. Thermophilic fungi of tobacco. Tech. Bull. No. 211, North Carolina Agric. Exp. Sta., Raleigh, NC. Tansey, M. R., 1971. Agar diffusion assay of cellulolytic ability of thermophilic fungi. Arch. Microbiol. 7 7 : 1 - 1 1 .