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Quantitative estimation of chitinase and several other enzymes in the fungus Beauveria bassiana
JOURNAL
OF
INVERTEBRATE
PATHOLOGY
15, 34-42
( 1970)
Quantitative Estimation of Chitinase and Several Other Enzymes in the Fungus Beuuveriu bassiuna J. LEOPOLD Lachema, lnstitute
1. FuMk’s
Works,
of Entomology,
AND ANNA Kazdjov,
and
Czechoslovakian
SAMSI~~KOVA Department Academy
of Insect of Science,
Pathology, Prague
6,
Czechoslovakia Received
May
27, 1969
A strain of the entomophagous fungus Beauveria bassiana was cultivated under stationary and submersed conditions. In the mycelium of stationary cultures of varying age and in culture medium of a 3-day-old submersed culture, the presence of chitinase, cellulase, proteases, and lipase was ascertained. Beauveria bussiana is capable of liberating these enzymes into the medium. Chitinase, of the strain of Beauveria bassiana tested, is a constitutive enzyme. Proteases split peptone and gelatine but not casein. The highest activity of chitinase and cellulase is found in 4-to-5-day-old mycelia, while the maximum amounts of proteases and lipase are formed after a 6-to7-day period of cultivation of the fungus.
chitin. We noted also the presence of cellulase, lipase, and the proteolytic enzymes.
INTRODUCTION
The fact that entomogenous fungi produce enzymes that dissolve chitin and enable the fungus to penetrate into the body MATERIALS AND METHODS of the insect has been coniirmed by several Fungus. Trials were performed with a investigators (Huber, 1958; Claus, 1961; strain of the entomogenous fungus BeauGabriel, 1968). Up to now, chitinase and veria bassiana from the collection of the a number of other enzymes have been Department of Insect Pathology of the Indemonstrated in the entomogenous species: Beauveria bassiana, Metarrhizium aniso- stitute of Entomology of the Czechoslovakian Academy of Science. It was isolated pliae, and Cordyceps militaris (Huber, 1958) and recently in several species of the from a larva of the Colorado beetle, Leptinotarsa decemplineata. genus Entomophthora (Gabriel, 1968). Preparation of Cultures. For the experiIn the present study we investigated chiefly the quantitative relations of the en- ments performed, submersed cultures (inzyme chitinase in Beauveria bassiana,which oculated with aerial conidia) were cultivated is one of the best known and most common for 3 days on a reciprocal shaking machine entomogenous fungi. It is polyphagous, kills at a temperature of 27°C in a medium havmany kinds of insects, and is not limited to ing the following composition: glucose 2.5%, a specific host. According to our experience starch 2.5%, corn steep 2.0%, NaCl 0.5%, 1966). ( Cermakova and Sam?Zakova, 1960)) the and CaC03 0.2% (Sam&“rikovi, The cultures were filtered and the filtrate fungus, when attacking the larva of the Colorado beetle, Leptinotarsa decemlineata, used as an enzymatic preparation. grows into and through the soft skin of the Besides submersed cultures, stationary insect rather than the parts covered with cultures cultivated in the same medium at 34
ENZYMES
IN
the same temperature were also used. After the formation of a continuous mycelial layer (after 3 days, at the earliest), the mycelium was collected at 24-hr intervals, and processed into enzymatic preparations. For a possible induction of chitinase, the C- or N-source or both were replaced, in media for submersed as well as for stationary cultures, by colloidal chitin or by chitosan in quantities of 1 to 2.5%, or these components were added to the complete medium. Mycelia cultivated in an optimal medium were collected at 24-hr intervals for the preparation of enzymatic products. Enzymatic Preparations. The enzymatic preparations were either filtrates of submersed cultures of the fungus or mycelia that, after being washed in water and squeezed, were triturated with glass powder in a weight ratio of 1:3 and diluted with water to make 40 ml. Determination of Chitinuse. A. The substrate used was commercially produced pure chitin. It was brought into a colloidal state by dissolution in concentrated hydrochloric acid. hfter filtration through asbestos the solution was poured into ice water while being stirred. The chitin floccules obtained were filtered, washed with water to get a neutral reaction, and washed with ether and dried in a vacuum. Acetylglucosamine, developed as the result of the action of chitinase, was determined reductometrically according to Schoorl ( 1929). This method, prepared for the determination of glucose, fructose, lactose, and maltose, was extended to cover the determination of acetylglucosamine. In Fig. 1 are plotted for the purpose of comparison the values for glucose and acetylglucosamine in relation to the consumption of 0.1 N NaaSzOB. To carry out the enzymatic tests, 200 mg of chitin in 20 ml of water and 20 ml of citrate buffer at pH 5 were placed in Erlenmeyer flasks, with an addition of 40 ml of enzymatic solution and 1.5% of toluene-
35
&XutWiU
30.
/
g
.
20.
./ .
10.
/
10
20
./*I’
30
40
50
60
70
1. Typical curves for determination (g = glucose, n = N-acetylglucoseamine) according to Schoorl’s method. X axis: mg sugar; Y axis: ml of 0.1 N Na,S,O,. FIG.
80
of
sugar
of
the latter to prevent contamination. Closed flasks were incubated at a temperature of 33°C. A blind trial was made in the same way but with an enzymatic solution inactivated by a 15-min. submersion into a boiling water bath. At given time intervals, specimens were collected and filtered, and a quantity of 10 ml was always taken for the determination of acetylglucosamine according to the method developed by Schoorl. B. For the estimation of chitinase by the viscosimetric method (Tracey, 1955), a 1% solution of chitosan was prepared. This was accomplished by adding to the suspension of chitosan in water a few milliliters of glacial acetic acid, properly stirring all the while. The chitosan was almost completely dissolved and a viscous solution formed; after adding water to attain something less than the desired volume, the solution was dialyzed for 2 to 3 days until the excess of acetic acid was removed. With the expected final volume of 1 liter, there was dissolved in the solution 21.5 g of NaCl, 19 g of Naacetate, 3 ml I&O, and an additional 3.6 g of glacial acetic acid. (By dissolving the buffer in the solution of the substrate, un-
36
LEOPOLD AND SAM?&liKOVti
necessary dilution and reduction of viscosity were avoided.) With each 30 ml of chitosan solution we used 30 ml of enzymatic solution (inactivated in the control). Viscosity was measured in a Hoppler’s viscosimeter; the decrease of viscosity is a measure of enzymatic degradation of chitosan. C. Chromatographic determination. Colloidal chitin was incubated with an enzymatic preparation at a temperature of 33°C and at certain intervals specimens were taken after filtration together with N-acetylglucosamine as a standard, applied on chromatographic paper and developed in a mixture of butanol, ethanol, and water in a ratio of 4: 1:5. AgN03 was used for the detection. Determination of Celluluse A. Qualitative assay. Into agar medium containing NaN03 0.2 g, KH2P0, 0.1 g, KC1 0.05 g, MgS04 0.02 g, CaC12 0.01 g, and a yeast extract 0.05 g in 100 ml of water, 0.25% of pure cellulose in powder form was added and stirred. In places where the inoculated microorganism produces cellulase, the slide does not stain blue with chlorzinciodine. B. As substrate, carboxymethylcellulose (CMC), previously freed of all impurities by extraction using 80% ethyl alcohol was used. Three grams of pure carboxymethylcellulose were mixed with 30 ml of water and the mixture allowed to swell overnight. The subsequent day the solution was made up with water to 100 ml and after mixing was filtered through a crucible with sintered glass Gl. With the use of phosphoric acid, the solution was modified to pH 5.2; thereafter it was centrifuged for a period of 10 min. The supernatant, which had been stored in the refrigerator, was used as substrate. The enzymatic determination was made after mixing 45 ml of the substrate with 5 ml of the enzymatic preparation in a Hbppler’s viscosimeter at a temperature of 50°C.
Determination of Proteases. Twenty ml of the enzymatic solution was allowed to act on 1.5 g of peptone, gelatin, or sodium caseinate in 40 ml of water at pH 5 and a temperature of 35°C after the addition of 1.5% of toluene. The enzymatically liberated amino acids were determined in lo-ml specimens after the addition of 90 ml of 96% alcohol. Titration was effected with an alcoholic solution of 0.2 N KOH with phenolphthalein. One ml of 0.2 N KOH corresponds to 2.8 mg of NH2 nitrogen. The result of the blind trial (with heat-inactivated enzymatic solution) is obtained from these data. Determination of Lipase. To determine the presence of lipase, the stalagmometric method was used; this method is recommended (Rona and Michaelis, 1931) in instances of weak enzymatic activity. To 50 ml of buffered water solution (pH 7.5) saturated with tributyrin, 2 ml of an enzymatic solution was added. The degree of splitting of tributyrin due to increased surface tension was measured. RESULTS
Chit&se A. Stationary cultures. Between the 2nd and 12th day, mycelia were taken from the stationary cultures and processed intc enzymatic preparations that were then tested for chitinase activity. Preliminary assays performed at various pH values revealed that the optimal chitinase activity was at about pH 5. At pH 3.5 and pH 7.0 the enzyme was strongly inhibited. All assays of chitinase determination were therefore continued at pH 5. The enzymatic preparations were allowed to act for a period of 7 days on chitin, and their activity was determined by the amount of N-acetylglucosamine developed. The results may be seen in Fig. 2. The highest chitinase activity was found after 7 days of incubation in 4- to 5-day-old cultures. In
ENZYMES
120.
Beauveria
IN
37
.-.
100
. :
./’ \
.
60
%Om/ 40.
\ l .
\
20 .
‘-. 2
FIG. 2. Chitinase cultures of varying 10 ml of solution.
3
determination. age on chitin.
4
5
6
7
8
Action of Beauveria X axis: age of cultures
younger or older cultures, chitinase activity rapidly decreases. B. Submersed cultures. Culture media were tested for their content of chitinase; in cultures 24- to 48-hr. old no action of chitinase was observed; in a 3-day-old culture the enzyme was most active, in older cultures the activity greatly declined, and in cultures older than 5 days it could not be demonstrated. For this reason a 3-day culture medium was tested for the content of chitinase by the determination of N-acetylglucosamine (using Schoorl’s method) in the course of a I2-day action on chitin. The results can be seen in Fig. 3. Also, in this case the splitting of chitin was optimal between the 7th and 8th day of incubation; at earlier or later periods activity decreased. C. Viscosimetric determination. Viscosimetric determinations confirmed these findings. The maximum activity of chitinase (the difference in velocity of the fall of a ball between the initial time and after 15 min = 5.6 set) ensues, in both instances,
9
10
11
12
bassiana mycelial enzymatic preparations in days; Y axis: mg of N-acetylglucosamine
from in
after a 7-day incubation. In stationary cultures the maximum activity occurs after 4 to 5 days; in submersed cultures, this takes place after 3 days. D. Chromatographic determination. The reductometric analysis of a 3-day culture medium for the content of chitinase, previously mentioned, was supplemented by a chromatographic analysis. In conformity with the reductometric analysis, it was found that the most intensive spot of Nacetylglucosamine occurred after a 7-day incubation of chitin with the cultivation medium (Fig. 4). E. Trials with chitinase induction. Both in the stationary and in the submersed culture, chitin was tested as a generator of chitinase formation. If in the culture medium the source of carbon or nitrogen was replaced by chitin, no increase in the enzyme formation was observed. When chitin was added to the complete medium, there was no evidence that the enzymatic activity of chitinase was increased. Nor could the
38
LEOPOLD
AND
SAhf.!&iKOVA
FIG. 3. Chitinase determination. Action of culture medium of a S-day-old submersed culture chitin for a period of 12 days. X axis: period of enzymatic action in days; Y axis: mg of N-acetylglucosamine in 10 ml of solution,
effect of the presence of chitin in the culture medium be observed in the growth, development, and virulence of the fungus (the amount of growth checked by the weight of the dry matter), or of mycehum itself (as determined by its microscopic appearance and parallel infection tests performed on larvae of Galbria meZZoneze2Za). Cellulase A. Qualitative assays showed that during a 3-day period of cultivation the fungus Beauveria bassiana secretes cellulase into the agar medium. B. Viscometric determination using carboxymethylcellulose as a substrate was carried out by enzymatic preparations from 3-to-g-day-old mycelia grown in stationary cultures. Figure 5 shows curves of viscosity of several tests in which the enzymatic preparations had been active for a period
on
of 30 min. Most active cellulolytic preparations were obtained from 4-to-S-day-old cultures (Fig. 6). The culture medium of a 3-day submersed culture exhibited a somewhat higher cellulolytic activity than that of the stationary culture of the same age. Proteases A. Stationary cultures. Although sodium caseinate was not degraded by enzymatic preparations from mycelia of varying ages, peptone and gelatin were attacked (Table 1). The splitting of the substrate continued throughout the incubation period. Beauveria bassiana thus contains proteolytic enzymes splitting peptone and gelatin but not the enzyme-splitting casein. When comparing the splitting values (Fig. 7) after a 6-day incubation, when the maximum of splitting occurred, we found that gelatin
ENZYMES
IN
Beauueria
39
1'
16.
FIG. 4. Chromatogram of chitin splitting. (a) Splitting of chitin by a mycelial enzymatic preparation. (b) Standard N-acetylglucosamine.
was being split more intensively than peptone. Both substrates were most degraded by the 6-to-7-day-old mycelium. B. In the submersed culture a filtrate of a 3-day-old culture was again tested; even in this case caseinate was not degraded, but only peptone and gelatin-of these, peptone much worse (1.1-4.2-5.3 mg of NHZN) than gelatin (5.6-10.9-15.7 mg of NHzN).
Lipase Enzymatic preparations from 3-to-g-dayold mvcelia were again tested, this time for the nresence of Iinase. When we nbt the
56.
0
10
20
30
FIG. 5. Cellulase determination. Splitting carboxymethylcellulose by mycelial enzymatic preparations from 3-to-g-day-old cultures and culture medium of submersed culture ( = s). axis: period of action of cellulase in minutes; axis: period of fall of the viscosimetric ball seconds. Numbers (3-9) = age of cultures days.
of a X Y in in
percentage of tributyrin degradation (stalagmometric measurements for a period of 45 min) against the age of the culture, we obtain the curve shown in Fig. 8. From the 3rd to the 5th day, activitv of
40
LEOPOLD
AND
Y ” I SAMSINAKOVii
20.
15. ‘-. \
x IO.
f
--•A,
‘5 -
3
4
5
6
7
8
9
FIG. 6. Cellulase determination. Splitting of carboxymethylcellulose by mycelial enzymatic prepcultures; Y axis: time difference of arations from cultures of different ages. X axis: age of stationary fall of a ball in seconds between the start and the end of the test (time period O-30 miu); x: submersed trial (culture medium after 3 days).
TABLE SPLITTING
ENZYMATIC CULTURES MG
AFTER
THE
Age
AND
OF NH,
BY
FROM AGE
N IN
READING
OF
VALUE OF THE BLIND TRIAL).
Incubation enzymatic
of culture,
GELATIN
PREPARATIONS OF VARYING
(INCREASE
lipases falls, but then it rapidly rises, achieving a maximum in the 7-day-old mycelium only to rapidly fall again. In the filtrate of a 3-day submersed culture, the lipolytic activity is as low as in a Sday-old least active mycelium of surface culture.
1
OF PEPTONE
period with preparations
DISCUSSION
2
4
6
Substrate
days
days
days
peptone gelatin
7.0 3.1 1.4 2.8 7.3 8.4 7.3 7.8 9.2 14.6 8.4 12.6 6.4 10.6
7.8 5.0 2.8 4.2 7.3 13.4 12.9 10.3 11.2 16.8 10.1 16.2 10.1 15.7
10.6 8.1 3.4 5.0 8.4 20.4 15.4 26.2 15.4 26.3 11.2 22.4 11.2 20.2
peptone gelatin peptone gelatin peptone gelatin peptone gelatin peptone gelatin peptone gelatin
When an insect is attacked by entomogenous fungi, the parasite is assumedto have invaded the body of its host by producing suitable enzymes by which it disintegrates the body surface of its victim. Because in the majority of cases penetration through the chitin shell of insects is involved, our attention was drawn chiefly to the ability of the parasite to produce the enzyme chitinase. The strain of the fungus under study forms chitinase both in stationary and submersed cultures and liberates it into the environment, which means that it is an extracellular enzyme. Huber (1958) assumes that chitin formation takes place chiefly in
ENZYMES IN Beuuveria
41
30 t
I 3 FIG. ‘7. Determination preparations from cultures ml of specimen,
4
5
of proteases. of different
6
!
8
9
Splitting of peptone (p) and gelatin (g) by mycelial enzymatic ages. X axis: age of culture in days; Y axis: mg of NH, N in 10
40 -
30.
20
10.
. \ Y
.
3
4
\
.Y
5
6
!
8
9
FIG. 8. Lipase determination. Splitting of tributyrin by mycelial enzymatic preparations tures of different ages. X axis: age of cultures in days; Y axis: degree of splitting of tributyrin cent; x: submersed culture (culture medium 3 days old).
the shoots of germinating conidia and in the “tops” or “ends” of hyphae. In our stationary cultures the highest chitinase activity was observed in cultures already fully developed. The question whether chitinase of the fungus Beauveria bassiuna is a constitutive or adaptive enzyme cannot be an-
from culin per-
swered in only a general way, because while Huber (1958) characterizes it as a constitutive enzyme, Gabriel (1968) refers to it as an adaptive enzyme. We classify chitinase of our strain of Beauveria bassiana as a constitutive enzyme. We believe that whether chitinase belongs to one group or
42
LEOPOLD
AND
the other depends on the specifity of the strain of the respective fungus. As already mentioned in the Introduction, Beauveria bassiana is capable of penetrating even through the body surface of the host organism in the structure of which chitin does not participate ( CermCkovL and SamGiiikovb, 1960). For this reason the tests were extended to include the demonstration of the presence of other enzymes. Cellulase, proteolytic enzymes, and lipase were demonstrated. While the activity of cellulase was highest after the 4th or 5th day of cultivation of the fungus, proteases and lipase were most active in 6-to-7-dayold cultures. As results of tests with culture media of submersed cultures show, the fungus Beauveria bassiana can also liberate cellulase, proteases, and lipase into the culture medium. The description of the enzymatic mechanism of the action of the entomogenous fungus on insects will be the subject of another study.
SAMh+iKOVti REFERENCES CLAUS, L. 1961. Untersuchungen iiber die Chitinasewirkung des insektentotenden Pilzes Beuuveriu bassiana ( BaIs. ) Vuill. Arch. Mikrobid., 40, 1746. GERM~OV~~, A., AND SAMSIUKOVA, A. 1960. Uber den Mechanismus des Durchdringens des P&es Beauveria bassiunu ( Bals. ) Vuill. in die Larve von Leptinotarsa decemlineata Say. Csl. Purasitologie, 7, 231-236. GABRIEL, R. P. 1968. Enzymatic activities of some entomophthorous fungi. J. Invertebrate Pathol., 11, 70-81. HUBER, J. 1958. Untersuchungen zur Physiologie insektentiitender Pilze. Arch. Mikrobiol., 29, 257-276. RONA, P., AND MICHAELIS, L. 1931. “Praktikurn der Physiologischen Chemie.” Vol. I. Berlin. SAMSIGAKOV& A. 1966. Growth and sporulation of submerged cultures of the fungus Beauveria bassiunu in various media. j. Invertebrate Pathol., 8, 395400. SCHOORL, N. 1929. Zuckertitration. 2. Untersuch. Lebensmittel, 57, 566-568. TRACEY, M. mycetes.
V.
1955. Biochem.
Chitinase in some I., 61, 579-586.
basidio-
OF
INVERTEBRATE
PATHOLOGY
15, 34-42
( 1970)
Quantitative Estimation of Chitinase and Several Other Enzymes in the Fungus Beuuveriu bassiuna J. LEOPOLD Lachema, lnstitute
1. FuMk’s
Works,
of Entomology,
AND ANNA Kazdjov,
and
Czechoslovakian
SAMSI~~KOVA Department Academy
of Insect of Science,
Pathology, Prague
6,
Czechoslovakia Received
May
27, 1969
A strain of the entomophagous fungus Beauveria bassiana was cultivated under stationary and submersed conditions. In the mycelium of stationary cultures of varying age and in culture medium of a 3-day-old submersed culture, the presence of chitinase, cellulase, proteases, and lipase was ascertained. Beauveria bussiana is capable of liberating these enzymes into the medium. Chitinase, of the strain of Beauveria bassiana tested, is a constitutive enzyme. Proteases split peptone and gelatine but not casein. The highest activity of chitinase and cellulase is found in 4-to-5-day-old mycelia, while the maximum amounts of proteases and lipase are formed after a 6-to7-day period of cultivation of the fungus.
chitin. We noted also the presence of cellulase, lipase, and the proteolytic enzymes.
INTRODUCTION
The fact that entomogenous fungi produce enzymes that dissolve chitin and enable the fungus to penetrate into the body MATERIALS AND METHODS of the insect has been coniirmed by several Fungus. Trials were performed with a investigators (Huber, 1958; Claus, 1961; strain of the entomogenous fungus BeauGabriel, 1968). Up to now, chitinase and veria bassiana from the collection of the a number of other enzymes have been Department of Insect Pathology of the Indemonstrated in the entomogenous species: Beauveria bassiana, Metarrhizium aniso- stitute of Entomology of the Czechoslovakian Academy of Science. It was isolated pliae, and Cordyceps militaris (Huber, 1958) and recently in several species of the from a larva of the Colorado beetle, Leptinotarsa decemplineata. genus Entomophthora (Gabriel, 1968). Preparation of Cultures. For the experiIn the present study we investigated chiefly the quantitative relations of the en- ments performed, submersed cultures (inzyme chitinase in Beauveria bassiana,which oculated with aerial conidia) were cultivated is one of the best known and most common for 3 days on a reciprocal shaking machine entomogenous fungi. It is polyphagous, kills at a temperature of 27°C in a medium havmany kinds of insects, and is not limited to ing the following composition: glucose 2.5%, a specific host. According to our experience starch 2.5%, corn steep 2.0%, NaCl 0.5%, 1966). ( Cermakova and Sam?Zakova, 1960)) the and CaC03 0.2% (Sam&“rikovi, The cultures were filtered and the filtrate fungus, when attacking the larva of the Colorado beetle, Leptinotarsa decemlineata, used as an enzymatic preparation. grows into and through the soft skin of the Besides submersed cultures, stationary insect rather than the parts covered with cultures cultivated in the same medium at 34
ENZYMES
IN
the same temperature were also used. After the formation of a continuous mycelial layer (after 3 days, at the earliest), the mycelium was collected at 24-hr intervals, and processed into enzymatic preparations. For a possible induction of chitinase, the C- or N-source or both were replaced, in media for submersed as well as for stationary cultures, by colloidal chitin or by chitosan in quantities of 1 to 2.5%, or these components were added to the complete medium. Mycelia cultivated in an optimal medium were collected at 24-hr intervals for the preparation of enzymatic products. Enzymatic Preparations. The enzymatic preparations were either filtrates of submersed cultures of the fungus or mycelia that, after being washed in water and squeezed, were triturated with glass powder in a weight ratio of 1:3 and diluted with water to make 40 ml. Determination of Chitinuse. A. The substrate used was commercially produced pure chitin. It was brought into a colloidal state by dissolution in concentrated hydrochloric acid. hfter filtration through asbestos the solution was poured into ice water while being stirred. The chitin floccules obtained were filtered, washed with water to get a neutral reaction, and washed with ether and dried in a vacuum. Acetylglucosamine, developed as the result of the action of chitinase, was determined reductometrically according to Schoorl ( 1929). This method, prepared for the determination of glucose, fructose, lactose, and maltose, was extended to cover the determination of acetylglucosamine. In Fig. 1 are plotted for the purpose of comparison the values for glucose and acetylglucosamine in relation to the consumption of 0.1 N NaaSzOB. To carry out the enzymatic tests, 200 mg of chitin in 20 ml of water and 20 ml of citrate buffer at pH 5 were placed in Erlenmeyer flasks, with an addition of 40 ml of enzymatic solution and 1.5% of toluene-
35
&XutWiU
30.
/
g
.
20.
./ .
10.
/
10
20
./*I’
30
40
50
60
70
1. Typical curves for determination (g = glucose, n = N-acetylglucoseamine) according to Schoorl’s method. X axis: mg sugar; Y axis: ml of 0.1 N Na,S,O,. FIG.
80
of
sugar
of
the latter to prevent contamination. Closed flasks were incubated at a temperature of 33°C. A blind trial was made in the same way but with an enzymatic solution inactivated by a 15-min. submersion into a boiling water bath. At given time intervals, specimens were collected and filtered, and a quantity of 10 ml was always taken for the determination of acetylglucosamine according to the method developed by Schoorl. B. For the estimation of chitinase by the viscosimetric method (Tracey, 1955), a 1% solution of chitosan was prepared. This was accomplished by adding to the suspension of chitosan in water a few milliliters of glacial acetic acid, properly stirring all the while. The chitosan was almost completely dissolved and a viscous solution formed; after adding water to attain something less than the desired volume, the solution was dialyzed for 2 to 3 days until the excess of acetic acid was removed. With the expected final volume of 1 liter, there was dissolved in the solution 21.5 g of NaCl, 19 g of Naacetate, 3 ml I&O, and an additional 3.6 g of glacial acetic acid. (By dissolving the buffer in the solution of the substrate, un-
36
LEOPOLD AND SAM?&liKOVti
necessary dilution and reduction of viscosity were avoided.) With each 30 ml of chitosan solution we used 30 ml of enzymatic solution (inactivated in the control). Viscosity was measured in a Hoppler’s viscosimeter; the decrease of viscosity is a measure of enzymatic degradation of chitosan. C. Chromatographic determination. Colloidal chitin was incubated with an enzymatic preparation at a temperature of 33°C and at certain intervals specimens were taken after filtration together with N-acetylglucosamine as a standard, applied on chromatographic paper and developed in a mixture of butanol, ethanol, and water in a ratio of 4: 1:5. AgN03 was used for the detection. Determination of Celluluse A. Qualitative assay. Into agar medium containing NaN03 0.2 g, KH2P0, 0.1 g, KC1 0.05 g, MgS04 0.02 g, CaC12 0.01 g, and a yeast extract 0.05 g in 100 ml of water, 0.25% of pure cellulose in powder form was added and stirred. In places where the inoculated microorganism produces cellulase, the slide does not stain blue with chlorzinciodine. B. As substrate, carboxymethylcellulose (CMC), previously freed of all impurities by extraction using 80% ethyl alcohol was used. Three grams of pure carboxymethylcellulose were mixed with 30 ml of water and the mixture allowed to swell overnight. The subsequent day the solution was made up with water to 100 ml and after mixing was filtered through a crucible with sintered glass Gl. With the use of phosphoric acid, the solution was modified to pH 5.2; thereafter it was centrifuged for a period of 10 min. The supernatant, which had been stored in the refrigerator, was used as substrate. The enzymatic determination was made after mixing 45 ml of the substrate with 5 ml of the enzymatic preparation in a Hbppler’s viscosimeter at a temperature of 50°C.
Determination of Proteases. Twenty ml of the enzymatic solution was allowed to act on 1.5 g of peptone, gelatin, or sodium caseinate in 40 ml of water at pH 5 and a temperature of 35°C after the addition of 1.5% of toluene. The enzymatically liberated amino acids were determined in lo-ml specimens after the addition of 90 ml of 96% alcohol. Titration was effected with an alcoholic solution of 0.2 N KOH with phenolphthalein. One ml of 0.2 N KOH corresponds to 2.8 mg of NH2 nitrogen. The result of the blind trial (with heat-inactivated enzymatic solution) is obtained from these data. Determination of Lipase. To determine the presence of lipase, the stalagmometric method was used; this method is recommended (Rona and Michaelis, 1931) in instances of weak enzymatic activity. To 50 ml of buffered water solution (pH 7.5) saturated with tributyrin, 2 ml of an enzymatic solution was added. The degree of splitting of tributyrin due to increased surface tension was measured. RESULTS
Chit&se A. Stationary cultures. Between the 2nd and 12th day, mycelia were taken from the stationary cultures and processed intc enzymatic preparations that were then tested for chitinase activity. Preliminary assays performed at various pH values revealed that the optimal chitinase activity was at about pH 5. At pH 3.5 and pH 7.0 the enzyme was strongly inhibited. All assays of chitinase determination were therefore continued at pH 5. The enzymatic preparations were allowed to act for a period of 7 days on chitin, and their activity was determined by the amount of N-acetylglucosamine developed. The results may be seen in Fig. 2. The highest chitinase activity was found after 7 days of incubation in 4- to 5-day-old cultures. In
ENZYMES
120.
Beauveria
IN
37
.-.
100
. :
./’ \
.
60
%Om/ 40.
\ l .
\
20 .
‘-. 2
FIG. 2. Chitinase cultures of varying 10 ml of solution.
3
determination. age on chitin.
4
5
6
7
8
Action of Beauveria X axis: age of cultures
younger or older cultures, chitinase activity rapidly decreases. B. Submersed cultures. Culture media were tested for their content of chitinase; in cultures 24- to 48-hr. old no action of chitinase was observed; in a 3-day-old culture the enzyme was most active, in older cultures the activity greatly declined, and in cultures older than 5 days it could not be demonstrated. For this reason a 3-day culture medium was tested for the content of chitinase by the determination of N-acetylglucosamine (using Schoorl’s method) in the course of a I2-day action on chitin. The results can be seen in Fig. 3. Also, in this case the splitting of chitin was optimal between the 7th and 8th day of incubation; at earlier or later periods activity decreased. C. Viscosimetric determination. Viscosimetric determinations confirmed these findings. The maximum activity of chitinase (the difference in velocity of the fall of a ball between the initial time and after 15 min = 5.6 set) ensues, in both instances,
9
10
11
12
bassiana mycelial enzymatic preparations in days; Y axis: mg of N-acetylglucosamine
from in
after a 7-day incubation. In stationary cultures the maximum activity occurs after 4 to 5 days; in submersed cultures, this takes place after 3 days. D. Chromatographic determination. The reductometric analysis of a 3-day culture medium for the content of chitinase, previously mentioned, was supplemented by a chromatographic analysis. In conformity with the reductometric analysis, it was found that the most intensive spot of Nacetylglucosamine occurred after a 7-day incubation of chitin with the cultivation medium (Fig. 4). E. Trials with chitinase induction. Both in the stationary and in the submersed culture, chitin was tested as a generator of chitinase formation. If in the culture medium the source of carbon or nitrogen was replaced by chitin, no increase in the enzyme formation was observed. When chitin was added to the complete medium, there was no evidence that the enzymatic activity of chitinase was increased. Nor could the
38
LEOPOLD
AND
SAhf.!&iKOVA
FIG. 3. Chitinase determination. Action of culture medium of a S-day-old submersed culture chitin for a period of 12 days. X axis: period of enzymatic action in days; Y axis: mg of N-acetylglucosamine in 10 ml of solution,
effect of the presence of chitin in the culture medium be observed in the growth, development, and virulence of the fungus (the amount of growth checked by the weight of the dry matter), or of mycehum itself (as determined by its microscopic appearance and parallel infection tests performed on larvae of Galbria meZZoneze2Za). Cellulase A. Qualitative assays showed that during a 3-day period of cultivation the fungus Beauveria bassiana secretes cellulase into the agar medium. B. Viscometric determination using carboxymethylcellulose as a substrate was carried out by enzymatic preparations from 3-to-g-day-old mycelia grown in stationary cultures. Figure 5 shows curves of viscosity of several tests in which the enzymatic preparations had been active for a period
on
of 30 min. Most active cellulolytic preparations were obtained from 4-to-S-day-old cultures (Fig. 6). The culture medium of a 3-day submersed culture exhibited a somewhat higher cellulolytic activity than that of the stationary culture of the same age. Proteases A. Stationary cultures. Although sodium caseinate was not degraded by enzymatic preparations from mycelia of varying ages, peptone and gelatin were attacked (Table 1). The splitting of the substrate continued throughout the incubation period. Beauveria bassiana thus contains proteolytic enzymes splitting peptone and gelatin but not the enzyme-splitting casein. When comparing the splitting values (Fig. 7) after a 6-day incubation, when the maximum of splitting occurred, we found that gelatin
ENZYMES
IN
Beauueria
39
1'
16.
FIG. 4. Chromatogram of chitin splitting. (a) Splitting of chitin by a mycelial enzymatic preparation. (b) Standard N-acetylglucosamine.
was being split more intensively than peptone. Both substrates were most degraded by the 6-to-7-day-old mycelium. B. In the submersed culture a filtrate of a 3-day-old culture was again tested; even in this case caseinate was not degraded, but only peptone and gelatin-of these, peptone much worse (1.1-4.2-5.3 mg of NHZN) than gelatin (5.6-10.9-15.7 mg of NHzN).
Lipase Enzymatic preparations from 3-to-g-dayold mvcelia were again tested, this time for the nresence of Iinase. When we nbt the
56.
0
10
20
30
FIG. 5. Cellulase determination. Splitting carboxymethylcellulose by mycelial enzymatic preparations from 3-to-g-day-old cultures and culture medium of submersed culture ( = s). axis: period of action of cellulase in minutes; axis: period of fall of the viscosimetric ball seconds. Numbers (3-9) = age of cultures days.
of a X Y in in
percentage of tributyrin degradation (stalagmometric measurements for a period of 45 min) against the age of the culture, we obtain the curve shown in Fig. 8. From the 3rd to the 5th day, activitv of
40
LEOPOLD
AND
Y ” I SAMSINAKOVii
20.
15. ‘-. \
x IO.
f
--•A,
‘5 -
3
4
5
6
7
8
9
FIG. 6. Cellulase determination. Splitting of carboxymethylcellulose by mycelial enzymatic prepcultures; Y axis: time difference of arations from cultures of different ages. X axis: age of stationary fall of a ball in seconds between the start and the end of the test (time period O-30 miu); x: submersed trial (culture medium after 3 days).
TABLE SPLITTING
ENZYMATIC CULTURES MG
AFTER
THE
Age
AND
OF NH,
BY
FROM AGE
N IN
READING
OF
VALUE OF THE BLIND TRIAL).
Incubation enzymatic
of culture,
GELATIN
PREPARATIONS OF VARYING
(INCREASE
lipases falls, but then it rapidly rises, achieving a maximum in the 7-day-old mycelium only to rapidly fall again. In the filtrate of a 3-day submersed culture, the lipolytic activity is as low as in a Sday-old least active mycelium of surface culture.
1
OF PEPTONE
period with preparations
DISCUSSION
2
4
6
Substrate
days
days
days
peptone gelatin
7.0 3.1 1.4 2.8 7.3 8.4 7.3 7.8 9.2 14.6 8.4 12.6 6.4 10.6
7.8 5.0 2.8 4.2 7.3 13.4 12.9 10.3 11.2 16.8 10.1 16.2 10.1 15.7
10.6 8.1 3.4 5.0 8.4 20.4 15.4 26.2 15.4 26.3 11.2 22.4 11.2 20.2
peptone gelatin peptone gelatin peptone gelatin peptone gelatin peptone gelatin peptone gelatin
When an insect is attacked by entomogenous fungi, the parasite is assumedto have invaded the body of its host by producing suitable enzymes by which it disintegrates the body surface of its victim. Because in the majority of cases penetration through the chitin shell of insects is involved, our attention was drawn chiefly to the ability of the parasite to produce the enzyme chitinase. The strain of the fungus under study forms chitinase both in stationary and submersed cultures and liberates it into the environment, which means that it is an extracellular enzyme. Huber (1958) assumes that chitin formation takes place chiefly in
ENZYMES IN Beuuveria
41
30 t
I 3 FIG. ‘7. Determination preparations from cultures ml of specimen,
4
5
of proteases. of different
6
!
8
9
Splitting of peptone (p) and gelatin (g) by mycelial enzymatic ages. X axis: age of culture in days; Y axis: mg of NH, N in 10
40 -
30.
20
10.
. \ Y
.
3
4
\
.Y
5
6
!
8
9
FIG. 8. Lipase determination. Splitting of tributyrin by mycelial enzymatic preparations tures of different ages. X axis: age of cultures in days; Y axis: degree of splitting of tributyrin cent; x: submersed culture (culture medium 3 days old).
the shoots of germinating conidia and in the “tops” or “ends” of hyphae. In our stationary cultures the highest chitinase activity was observed in cultures already fully developed. The question whether chitinase of the fungus Beauveria bassiuna is a constitutive or adaptive enzyme cannot be an-
from culin per-
swered in only a general way, because while Huber (1958) characterizes it as a constitutive enzyme, Gabriel (1968) refers to it as an adaptive enzyme. We classify chitinase of our strain of Beauveria bassiana as a constitutive enzyme. We believe that whether chitinase belongs to one group or
42
LEOPOLD
AND
the other depends on the specifity of the strain of the respective fungus. As already mentioned in the Introduction, Beauveria bassiana is capable of penetrating even through the body surface of the host organism in the structure of which chitin does not participate ( CermCkovL and SamGiiikovb, 1960). For this reason the tests were extended to include the demonstration of the presence of other enzymes. Cellulase, proteolytic enzymes, and lipase were demonstrated. While the activity of cellulase was highest after the 4th or 5th day of cultivation of the fungus, proteases and lipase were most active in 6-to-7-dayold cultures. As results of tests with culture media of submersed cultures show, the fungus Beauveria bassiana can also liberate cellulase, proteases, and lipase into the culture medium. The description of the enzymatic mechanism of the action of the entomogenous fungus on insects will be the subject of another study.
SAMh+iKOVti REFERENCES CLAUS, L. 1961. Untersuchungen iiber die Chitinasewirkung des insektentotenden Pilzes Beuuveriu bassiana ( BaIs. ) Vuill. Arch. Mikrobid., 40, 1746. GERM~OV~~, A., AND SAMSIUKOVA, A. 1960. Uber den Mechanismus des Durchdringens des P&es Beauveria bassiunu ( Bals. ) Vuill. in die Larve von Leptinotarsa decemlineata Say. Csl. Purasitologie, 7, 231-236. GABRIEL, R. P. 1968. Enzymatic activities of some entomophthorous fungi. J. Invertebrate Pathol., 11, 70-81. HUBER, J. 1958. Untersuchungen zur Physiologie insektentiitender Pilze. Arch. Mikrobiol., 29, 257-276. RONA, P., AND MICHAELIS, L. 1931. “Praktikurn der Physiologischen Chemie.” Vol. I. Berlin. SAMSIGAKOV& A. 1966. Growth and sporulation of submerged cultures of the fungus Beauveria bassiunu in various media. j. Invertebrate Pathol., 8, 395400. SCHOORL, N. 1929. Zuckertitration. 2. Untersuch. Lebensmittel, 57, 566-568. TRACEY, M. mycetes.
V.
1955. Biochem.
Chitinase in some I., 61, 579-586.
basidio-