- Email: [email protected]
Chitinase production by an Arthrobacter sp. lysing cells of Fusarium roseum
Soil Biol. Biochem.
Vol. 8, pp. 23-28.
Pergamon
Press
1976. Printed
in Great
Rritain.
CHITINASE PRODUCTION BY AN ARTHROBACTER LYSING CELLS OF FUSARIUM ROSEUM
SP.
R. F. MORRISSEY*, E. P. DUGAN and J. S. KOTHS Plant
Science Department.
University
(Accepted
of Connecticut
Storrs, Conn. 06268, U.S.A.
30 May 1975)
Summary-An Arthrobacter sp. which actively lysed Fusarium roseurn was found to liberate chitinase (E.C. 3.2-1.14, chitin glycanohydrolase), an enzyme essential for the hydrolysis of chitin, a major component of fusarial hyphal wails. Factors involved in the production of chitinase were investigated by modifying culture conditions and assaying for enzyme activity. Production occurred on colloidal chitin as well as on native chitin supplemented with yeast extract or peptone. Enzyme production paralleled growth; liberation of enzyme took place during the log phase with the maximum yield being obtained at the stationary phase. Addition of the non-ionic surfactant, polyoxyethylene sorbitan monooleate (Tween 80) increased enzyme yield. An inverse relationship was found between the amount of enzyme produced and the quantity of n-acetyl-glucosamine liberated. The enzyme was generally not produced when grown on various other carbohydrates. These findings suggest that chitinase is inducible and that chitin breakdown is regulated by a repressor-inducer mechanism. Initial hydrolysis rates of colloidal chitin were proportional to the concentration of chitinase used. Optimal pH and temperature for enzyme activity were 4.9 and 50°C. respectively. Purification of the chitinase was obtained by (NH&SO, precipitation followed by DEAE-cellulose and Sephadex chromatography, achieving a l2-fold increase in specific activity. INTRODUCTION
extract 0.2 g, mineral salts solution I I. The mineral salts solution was prepared from stock solutions to give the following final concentration per liter: KHzPOd, 4g; K?HPO,, 3g; MgS04.7H>O, 0.2g; HIB02, 5.6 mg: MnClz 4H20, 3.7 mg; CoClz 6Hz0, 0.7 mg; CuS04. 5Hz0, 0.4 mg; ZnS04. 7H20. 0.5 mg; NaMoO, 2H:O. I.5 mg; Fe-DTPA I.0 mg; and CaCI:, IO mg. The pH after autoclaving was cu. 6.5. Stock cultures were transferred monthly, incubated for 72 h at 25°C and stored at 4°C. The inoculum for all experiments was prepared from an overnight broth culture incubated at room temperature. The medium contained 0.1% glucose and 0.05% yeast extract in mineral salts solution. Glucose was autoclaved separately and added as a sterile 1% solution. Flasks were inoculated to an initial concentration of approximately 3 x IO’ cells/ml total count. After the required incubation, the bacterial cells were sedimented at 10,000 g for 20 min at room temperature. The cell-free supernatant was either cooled to 4°C or frozen at -5°C for future use. Unless otherwise stated, all cultures were incubated on a rotary shaker at 22-23°C.
In recent years the study of microbial interactions has led to increased interest in the use of microorganisms for the biological control of soilborne plant pathogens. This native method of reducing the incidence of plant disease obviates the need for chemical agents frequently linked with environmental contamination. Koths and Gunner (1967) utilized the principle of biological control for the protection of carnations against Fusarium stem rot. Protection was afforded through the introduction of a predaceous Arthrohacter sp. isolated from the carnation rhizosphere. This bacterium actively lysed living Fusarium mycelium in liquid media. Fundamental to the lytic phenomenon is the liberation by the bacterium of various enzymes which degrade the fungal cell wall. One such enzyme is chitinase. Chitinase hydrolyzes chitin, the p 1-4 linked polymer of n-acetyl-glucosamine, an important structural constituent of Fusarium sp. and many other fungi. The present investigation is concerned with the various parameters governing the production, properties and purification of the chitinase enzyme liberated by an Arthrobacter sp. MATERIALS
Preparation of substrates Unbleached crude chitin (1006) was washed alternately for 24 h at a time with 2 I 1 N NaOH and then 2 I 1 N HCI, for a total of eight washings. The suspension was held at 4°C during washings. The chitin was then rinsed 5 times with distilled water, twice with 95% ethanol and once with acetone. This treatment resulted in clean, white flakes. Colloidal chitin was prepared by the method of Lingappa and Lockwood (1962). Dry weights were determined by passing known volumes of colloidal chitin suspension through preweighed cellulose acetate filters (0.45 pm) and drying at 60°C. One drop of CHCI, was added per 100 ml and the stock
AND METHODS
The bacterium (P35) employed throughout this investigation was isolated from the rhizosphere of carnations (Dianthus caryophyllus) and used in studies of biological control of Fusarium roseum (Koths and Gunner, 1967). Subsequent identification placed the organism in the genus Arfhrohacter. Culture conditions Arthrobacter P-35 was maintained the following composition: glucose * Present address: N.J. 08903, U.S.A.
Johnson &Johnson.
on slants of 1 .O g, yeast New Brunswick,
23
24
R.
F.
MORRISSEY et al.
suspension was stored at 4°C. Before use the CHCI, was removed by placing the suspension in a boiling water bath until no odor of CHCI, could be detected. Fusarium roseam was grown in 3 1. quantities in a medium consisting of 10 g sucrose in 1 1. mineral salts. Aeration was achieved by sparging the culture with sterile compressed air. After incubation at room temperature for 4-5 days, most of the conidia were removed by washing the hyphae on a 79mesh/cm sieve. The fungus was then killed by immersion in 95% ethanol. The killed mycelium was ground in a blender, washed with distilled HZ0 and resuspended to provide a standard suspension. The dry weight of the mycelium was c. IO mg/ml. Assay procedures To determine chitinase activity, l-0 ml colloidal chitin (IO mglml) and 3.0 ml of 50 mM citrate buffer (pH 5.05) were incubated with I.0 ml of appropriately diluted cell-free culture supernatant. After 0 and 60 min incubation in a water bath at 37”C, I .O ml was pipetted into a screw cap tube containing 1 .O ml distilled H20 and the reaction stopped by placing the tube in a boiling water bath for 15 min. The tubes were cooled in tap water, the contents transferred to centrifuge tubes and spun down to remove residual chitin. The amount of n-acetylglucosamine present in I.0 ml of the supernatant was determined (Reissig, Strominger and Leloir, 1955) using a standard curve for n-acetylglucosamine. A milliunit of enzyme activity (mu) is defined as the amount of enzyme required to release 1 micromole of n-acetyl-glucosamine under the described conditions. Protein was determined by the method of Lowry et nl. (1951) with crystalline bovine serum albumin fraction V as a standard. Bacterial counts were made by plating serial dilutions on glucose-yeast extract-mineral salts agar and incubating at 25°C for 3 days. ~ur~~cat~on of ck~tinase (a) (NH,), SOn prec~p~tati~~. A 4 day old culture of Artkrubacter P-35 grown on mineral salts medium with 0.05% peptone and 1.0% I6 mesh/cm chitin was freed of cellular debris by centrifugation at 10,OOOg. The pH of the supernatant was adjusted to 7.0 by the addition of 2 M H>HPO,. (NH& SO, was added to the supernatant and a 30-90% fraction was collected on a Celite pad over filter paper placed in a Buchner funnel. The precipitate was eluted from the Celite by dissolving in 5 mM Na phosphate, pH 7.0. Since some of the enzyme remained absorbed at this pH, the Celite was rewashed using a second phosphate buffer having a pH of 8.9. The Celite was separated by centrifugation. The (NH&SO4 was removed by passing the chitinase solution through a G-25 Sephedex column. (b) DEAE-cellulose chromatography. The DEAE-cellulose columns were thoroughly equilibrated with 10 mM Na phosphate buffer pH 8.9. The pH of the crude chitinase solution was adjusted to 8.9 with 0.1 M NaOH. After passing through the columri the eluted chitinase was adjusted to pH 5.0 with 2 M Na H?P04.
(c) G-75 Sepkade.~ ckr(}~zata~rupky. The enzyme solution was concentrated by dialysis against Aquacidc (Calbiochem). After packing the column according to the manufacturer’s instructions, the enzyme solution was applied in 4 ml quantities and eluted with 50 mM citrate buffer, pH 5.0. RESULTS AND DISCUSSION
Production of ckitinuse The effect of various carbon sources on the appearance of chitinase in cell-free culture supernatants of Artkrohacter P-35 showed (Table I) that the yield of chitinase produced on colloidal chitin was over twice that of any non-chitinous substrate. The monomer of chitin, ~~-acetyl-glucosamine, failed to produce enzyme at both concentrations tested. Glucosamine HCl yielded some enzyme. Growing the organism on disaccharides (lactose and cellobiose) containing glycosidic linkages similar to chitin (beta-1,4) resulted in no enzyme production. Substantial chitinase production occurred when the organism was grown on yeast extract or peptone. Since the exact nature of these substances has not been chemically defined, interpretation of this data is difficult. However, because “yeast extract” is a water soluble extract of dried yeast cells. the possibility exists that soluble oligomers of yeast wall fractions could be present and that these induce chitinase production. Although some acidic metabolites were produced (Table I). the pH of the media did not fall below 5.65. well within the growth range tolerated by the bacterium. Unknown impurities in reagents used in the preparation of the media may have stimulated enzyme formation. Mandels et al. (1962) found that sophorose present in reagent grade glucose was a powerful inducer of cellulase in Trickodermu viride. When Artkr~~~~acterP-35 was grown on colloidal chitin, chitinase was liberated during the exponential phase (Fig. I), suggesting that the enzyme is extracellular. Grinding chitin flakes in a Wiley mill or adding Tween 80 to the culture media substantially increased chitinase production (Table 2). Production of enzyme on 16 mesh milled chitin was greater Table I. EfFect of various carbon sources on chitinasc production after 72 h incubation
Chitinase
l-.
NO. OF P35
0-o
PROTEIN
&--A
CHITINASE
production
by a fusarolytic
Arthrobacter
25
CELLS
8-
6)r- -
TIME
Fig.
Table
(h
1
1. Liberation of chitinase during growth on colloidal
DAYS)
by Arthrobacter chitin (4 mg/ml).
2. Effect of modification of chitin production after 94 h incubation
P-3.5
Fig. 2. Accumulation milled chitin
mesh). The
medium contained 0.05% salts solution.
increased
until Unlike colloidal completely digested
on chitinase
was not experiment.
at the
end
chitin of the
chitin did not even incubation. from to increased from 225 to 300 milliunits, while caused a doubling enzyme concentration. chitin was raised 2.0%, however, enzyme production occurred. The relationship enzyme production. 2 chitin enzyme
than that of colloidal chitin (without Tween SO). Greater accessibility to enzymatic attack (and therefore greater enzyme production) probably resulted from increasing the surface area of the flakes by milling. A similar stimulatory effect was obtained by the addition of Tween 80. Large increases were observed in the case of chitin flakes (4 fold) and F~~auilrrn mycelium (over 3 fold). N-deacetylation of chitin to form chitosan was sufficient to prevent enzyme production. Monreal and Reese (1969) also reported the inability of chitosan to induce chitinase in a strain of Serratia marcescens. The decrease in chitinase production on 24 mesh/cm chitin is attributed to the tendency of the finer particles to adhere to the walls of the culture flasks. Addjtion of Tween 80 overcame this problem and resulted in the highest chitinase production. The formation of chitinase resulting from growth on F. roseurn mycelium indicates that the fungus contains substances necessary for the production of the chitin in hyphal walls 1965). The high of enzyme chitin compared other carbon (Table 1) and the fact that modification chitin to chitosan prevents chitinase (Table 2) indicates enzyme is inducible.
chitin stable in culture.
enzyme
milled was extremely
during 7 days of incubation.
After
chitin
is
presented milled
chitin
caused
an
increase When
slight increase the concentration increase
(Fig. 3a). 2.0%, a enzyme
was
greatly
de-
pressed. chitin was employed growth
substrate
(Fig.
products.
Other
uptake hydrolytic
chitin
that when readily added to cultures, repression occurs. Before enzyme prothese readily utilizable first be consumed (Mandels and Reese, 1960; and Parrish, 1969; Monreal Reese, 1969). these experiments, enzyme production probably steady condition. enzyme production promotes chitin hydrolysis until a is reached where enzyme synthesis. point, the maximum concentration exist, The concentration chitin therefore effect on the rate of hydrolysate liberation. Table shows the effect adding X0 (sorbitan polyoxyethylene monooleate) yield of chitinase. Statistically significant
utilizable catabolic duction
have
of the
reports
w1
26
R. F. MORRISSEY et al.
0.6 2.4 I.2 3.0 1.8
200 *50 100 130 50
n
-b
n Llnll 0
20
0.45
MILLED
CHITIN
0
(%I
0.623
COLLOIDAL
1.25
CHITIN
(mg f ml
)
Fig. 3. Maximum amounts of chitinase and rz-acetyl-glucosamine liberated in cultures various concentrations of (a) milled chitin (I6 mesh) after 120 h incubation and (h) colloidal 70 h incubation. Table
3. Effect of the addition of Tween 80 on chitinase production after 120 h incubation
increases (P
b
80 t 60
40
m
O’ 0
F-H
Fig. 4. Effect
I ’
s mL
40 TEMPERATIJRE 20
of (a) pH and (b) temperature activity.
60 c“C,
0
’
80
on chitinase
containing chitin after
mately 25% of the maximum activity remained at pH 8.5 in 50 mM Tris-HCI buffer. The optimum temperature for the majority of chitinases has been reported to be 40°C (Jeuniaux, 1963). Monreal and Reese (1969) however, reported an optimum temperature of 50°C for the hydrolysis of colloidal chitin by S. marcescens. The data (Fig. 4b) show an optimum temperature at 50°C. with activity rapidly decreasing beyond this point. About 88% of the enzyme was inactivated after incubation for 60 min at 70°C. Routine enzyme assays were conducted at 37°C. The assignment of unit enzyme activity is based upon a linear relationship between the amount of nacetyl-glucosamine released by the hydrolysis and the concentration of the enzyme. The straight line relationship shown in Fig. 5a represents typical results found during this investigation. The proportionality indicates that the assay was free of inhibitory substances which could cause a decline in activity as the concentration of enzyme is increased. In each experiment requiring the assignment of enzyme units, a portion of the enzyme was tested to assure that the production of n-acetylglucosamine was linear with enzyme concentration over the range for unit activity. F&I-e 5b shows the effect of varying the concentration of colloidal chitin on the liberation of n-acetyl-glucosamine. The graph indicates that approximately 1.6-2.0 tig/ml of colloidal chitin are necessary to saturate the enzyme. This experiment was conducted using an undiluted sample of culture supernatant of high activity (0.380 PM n-acetylglucosamine liberated by I4 ml under standard conditions). Since standard enzyme assays used diluted samples, it follows that the 2.0 mg/ml concentration of colloidal chitin used in standard assays would always be sufficient to saturate. Purification of chitinase Several workers have used (NH&SO, precipitation of chitinases as an important part of their purification scheme. Berger and Reynolds (1958) using the culture supernatant from S. griseus obtained the greatest amount of chitinase with a salt saturation of 70% and a pH of 7.0. Skujins, Pukite and McLaren (1970), also working with 5’. griseus chitinase. used the same conditions appar-
$-
Chitinase production by a fusarolytic A~~lr~b~(~fer
27
loo-
0 0
60
/-
60 0
40-
/
/
-
RELATIVEDILUTION OFCULTURE SUPERNATANT
COLLOIDAL CHITIN (mg/mll
Fig. 5. Effect of (a) enzyme concentration and (b) substrate concentration n-acetyl-glucosamine
ently with good results. However, for the purification of Streptomyces antibioticus chitinase, Jeuniaux (1963) collected a 30-50% saturated fraction at a pH of 5-2. This followed adsorption and desorption from chitin. Monreal and Reese (1969) obtained high yields with a 40-80% fraction for the chitinase of S. marcescens. In our work the precipitate obtained at 30% saturation had relatively low specific activity and was discarded. The 30-90% fraction resulted in a 3 fold increase in specific activity. However, one-third of the total chitinase activity was lost with this procedure (Table 4). Skujins et a/. (1970) reported that at pH 8.9 S. griseus chitinase passed through a DEAF-cellulose column while other proteins and phenolic compounds remained in the column. However, with our Art~r~~acter chitinase, much of the enzyme became bound to the column resulting in a two-thirds loss in enzyme units and very little increase in specific activity. These results agree somewhat with the S. marcescens chitinase of Monreal and Reese (1969) which could not be recovered in good quantities from a DEAESephadex column. Skujins et al. (1970) used hydroxypapatite column chromatography to remove proteinase activity from their chitinase preparation. Several attempts to use this technique demonstrated that very little Arthrohacter chitinase could be eluted from the column and the use of hydroxyapatite was abandoned. Chitinase was separated from many other proteins on G-75 Sephadex. The effectiveness of the gel chromatography is illustrated by the elution profile (Fig. 6). Through this procedure a 3 fold increase in specific activity was achieved (Table 4). Table 4. Purification of chitinase
on the production
of
from colloidal chitin.
Fig. 6. Elution profile of chitinase from a G-75 Sephadex column. Fractions 20 through 27 were pooled. The final preparation
resulted
in specific supernatant.
compared
activity
in a 12 foId increase
with the culture
Acknowledgements-Scientific Contribution No. 608, Storrs Agricultural Experiment Station, The University of Connecticut, Storrs, Connecticut 06268, U.S.A. REFERENCES ARONSON J. M. (196.5)The celi wall. In The Fungi, Vol. I,
(G. C. Ainsworth and A. S. Sussman, Eds) pp. 49-76. Academic Press, New York. BERCERL. R. and REYNOLDSD. M. (1958) The chitinase system of a strain of ~treptomyces griseus. Riochem. hiophys. Acta 29, 522-534. JEUNIAUXC. (1963) Chitine et Chitinolyse. Masson et tie, Paris. KOTHSJ. S. and GUNNERH. B. (1967) Establishment of a rhizosphere microflora on carnation as a means of plant protection in steamed greenhouse soils. Proc. Am. Sot. hod. Sci. 91, 617-626. LOWRY 0. H., R~SEBROUGHN. J., FARR A. L. and RANDALLR. J. (1951) Protein measurement with the folin phenol reagnet. J. biol. Chem. 193, 265-275. LINCAPPAY. and LOCKWOOD J. L. (1962) Chitin media for selective isolation and culture of actinomycetes. Phyfopafho~o~y
52, 3 17-323.
MANDELS M., PARRISH F. W. and REESE E. T. (1962) Sophorose as an inducer of cellulase in Trichoderma viride. .I. Bact. 83, 400-408.
MANDELS M. and REESE E. T. (1960) Induction of cellulase in fungi by cellobiose. J. Bact. 79, 816-826. MONREALJ. and REESE E. T. (1969) The chitinase of Serratia marcescens. Can. J. Microbial. 15,689-696. POLLOCKM. R. (1962) Exoenzymes. In The Bacteria, Vol. IV (I. C. Gunsalus and R. Y. Stainer, Eds) 121-178. Academic Press, New York.
28
R. F. M~RRISSEY et al.
REESE E. T.. LOLA J. E. and PARRISH F. W. (1969) Modified substrates and modified products as inducers of carbohydrases. J. Bact. 100, 1151-1154. REISSIG J. R., STROMINGERJ. L. and LELOIR (1955) A modified calorimetric method for the estimation of ,I-acetylamino sugars. J. hiol. Chem. 217, 959-966.
REYNOLDS D. M. (1954) Exocellular chitinase from a streptomycete sp. J. gen. Microbial. 11, IX&159. SKUJINS J., PUKITE A. and MCLAREN A. D. (1970) Chitinase of Streptomyces sp.: purification and properties. Enzymologia 39, 353-370.
Vol. 8, pp. 23-28.
Pergamon
Press
1976. Printed
in Great
Rritain.
CHITINASE PRODUCTION BY AN ARTHROBACTER LYSING CELLS OF FUSARIUM ROSEUM
SP.
R. F. MORRISSEY*, E. P. DUGAN and J. S. KOTHS Plant
Science Department.
University
(Accepted
of Connecticut
Storrs, Conn. 06268, U.S.A.
30 May 1975)
Summary-An Arthrobacter sp. which actively lysed Fusarium roseurn was found to liberate chitinase (E.C. 3.2-1.14, chitin glycanohydrolase), an enzyme essential for the hydrolysis of chitin, a major component of fusarial hyphal wails. Factors involved in the production of chitinase were investigated by modifying culture conditions and assaying for enzyme activity. Production occurred on colloidal chitin as well as on native chitin supplemented with yeast extract or peptone. Enzyme production paralleled growth; liberation of enzyme took place during the log phase with the maximum yield being obtained at the stationary phase. Addition of the non-ionic surfactant, polyoxyethylene sorbitan monooleate (Tween 80) increased enzyme yield. An inverse relationship was found between the amount of enzyme produced and the quantity of n-acetyl-glucosamine liberated. The enzyme was generally not produced when grown on various other carbohydrates. These findings suggest that chitinase is inducible and that chitin breakdown is regulated by a repressor-inducer mechanism. Initial hydrolysis rates of colloidal chitin were proportional to the concentration of chitinase used. Optimal pH and temperature for enzyme activity were 4.9 and 50°C. respectively. Purification of the chitinase was obtained by (NH&SO, precipitation followed by DEAE-cellulose and Sephadex chromatography, achieving a l2-fold increase in specific activity. INTRODUCTION
extract 0.2 g, mineral salts solution I I. The mineral salts solution was prepared from stock solutions to give the following final concentration per liter: KHzPOd, 4g; K?HPO,, 3g; MgS04.7H>O, 0.2g; HIB02, 5.6 mg: MnClz 4H20, 3.7 mg; CoClz 6Hz0, 0.7 mg; CuS04. 5Hz0, 0.4 mg; ZnS04. 7H20. 0.5 mg; NaMoO, 2H:O. I.5 mg; Fe-DTPA I.0 mg; and CaCI:, IO mg. The pH after autoclaving was cu. 6.5. Stock cultures were transferred monthly, incubated for 72 h at 25°C and stored at 4°C. The inoculum for all experiments was prepared from an overnight broth culture incubated at room temperature. The medium contained 0.1% glucose and 0.05% yeast extract in mineral salts solution. Glucose was autoclaved separately and added as a sterile 1% solution. Flasks were inoculated to an initial concentration of approximately 3 x IO’ cells/ml total count. After the required incubation, the bacterial cells were sedimented at 10,000 g for 20 min at room temperature. The cell-free supernatant was either cooled to 4°C or frozen at -5°C for future use. Unless otherwise stated, all cultures were incubated on a rotary shaker at 22-23°C.
In recent years the study of microbial interactions has led to increased interest in the use of microorganisms for the biological control of soilborne plant pathogens. This native method of reducing the incidence of plant disease obviates the need for chemical agents frequently linked with environmental contamination. Koths and Gunner (1967) utilized the principle of biological control for the protection of carnations against Fusarium stem rot. Protection was afforded through the introduction of a predaceous Arthrohacter sp. isolated from the carnation rhizosphere. This bacterium actively lysed living Fusarium mycelium in liquid media. Fundamental to the lytic phenomenon is the liberation by the bacterium of various enzymes which degrade the fungal cell wall. One such enzyme is chitinase. Chitinase hydrolyzes chitin, the p 1-4 linked polymer of n-acetyl-glucosamine, an important structural constituent of Fusarium sp. and many other fungi. The present investigation is concerned with the various parameters governing the production, properties and purification of the chitinase enzyme liberated by an Arthrobacter sp. MATERIALS
Preparation of substrates Unbleached crude chitin (1006) was washed alternately for 24 h at a time with 2 I 1 N NaOH and then 2 I 1 N HCI, for a total of eight washings. The suspension was held at 4°C during washings. The chitin was then rinsed 5 times with distilled water, twice with 95% ethanol and once with acetone. This treatment resulted in clean, white flakes. Colloidal chitin was prepared by the method of Lingappa and Lockwood (1962). Dry weights were determined by passing known volumes of colloidal chitin suspension through preweighed cellulose acetate filters (0.45 pm) and drying at 60°C. One drop of CHCI, was added per 100 ml and the stock
AND METHODS
The bacterium (P35) employed throughout this investigation was isolated from the rhizosphere of carnations (Dianthus caryophyllus) and used in studies of biological control of Fusarium roseum (Koths and Gunner, 1967). Subsequent identification placed the organism in the genus Arfhrohacter. Culture conditions Arthrobacter P-35 was maintained the following composition: glucose * Present address: N.J. 08903, U.S.A.
Johnson &Johnson.
on slants of 1 .O g, yeast New Brunswick,
23
24
R.
F.
MORRISSEY et al.
suspension was stored at 4°C. Before use the CHCI, was removed by placing the suspension in a boiling water bath until no odor of CHCI, could be detected. Fusarium roseam was grown in 3 1. quantities in a medium consisting of 10 g sucrose in 1 1. mineral salts. Aeration was achieved by sparging the culture with sterile compressed air. After incubation at room temperature for 4-5 days, most of the conidia were removed by washing the hyphae on a 79mesh/cm sieve. The fungus was then killed by immersion in 95% ethanol. The killed mycelium was ground in a blender, washed with distilled HZ0 and resuspended to provide a standard suspension. The dry weight of the mycelium was c. IO mg/ml. Assay procedures To determine chitinase activity, l-0 ml colloidal chitin (IO mglml) and 3.0 ml of 50 mM citrate buffer (pH 5.05) were incubated with I.0 ml of appropriately diluted cell-free culture supernatant. After 0 and 60 min incubation in a water bath at 37”C, I .O ml was pipetted into a screw cap tube containing 1 .O ml distilled H20 and the reaction stopped by placing the tube in a boiling water bath for 15 min. The tubes were cooled in tap water, the contents transferred to centrifuge tubes and spun down to remove residual chitin. The amount of n-acetylglucosamine present in I.0 ml of the supernatant was determined (Reissig, Strominger and Leloir, 1955) using a standard curve for n-acetylglucosamine. A milliunit of enzyme activity (mu) is defined as the amount of enzyme required to release 1 micromole of n-acetyl-glucosamine under the described conditions. Protein was determined by the method of Lowry et nl. (1951) with crystalline bovine serum albumin fraction V as a standard. Bacterial counts were made by plating serial dilutions on glucose-yeast extract-mineral salts agar and incubating at 25°C for 3 days. ~ur~~cat~on of ck~tinase (a) (NH,), SOn prec~p~tati~~. A 4 day old culture of Artkrubacter P-35 grown on mineral salts medium with 0.05% peptone and 1.0% I6 mesh/cm chitin was freed of cellular debris by centrifugation at 10,OOOg. The pH of the supernatant was adjusted to 7.0 by the addition of 2 M H>HPO,. (NH& SO, was added to the supernatant and a 30-90% fraction was collected on a Celite pad over filter paper placed in a Buchner funnel. The precipitate was eluted from the Celite by dissolving in 5 mM Na phosphate, pH 7.0. Since some of the enzyme remained absorbed at this pH, the Celite was rewashed using a second phosphate buffer having a pH of 8.9. The Celite was separated by centrifugation. The (NH&SO4 was removed by passing the chitinase solution through a G-25 Sephedex column. (b) DEAE-cellulose chromatography. The DEAE-cellulose columns were thoroughly equilibrated with 10 mM Na phosphate buffer pH 8.9. The pH of the crude chitinase solution was adjusted to 8.9 with 0.1 M NaOH. After passing through the columri the eluted chitinase was adjusted to pH 5.0 with 2 M Na H?P04.
(c) G-75 Sepkade.~ ckr(}~zata~rupky. The enzyme solution was concentrated by dialysis against Aquacidc (Calbiochem). After packing the column according to the manufacturer’s instructions, the enzyme solution was applied in 4 ml quantities and eluted with 50 mM citrate buffer, pH 5.0. RESULTS AND DISCUSSION
Production of ckitinuse The effect of various carbon sources on the appearance of chitinase in cell-free culture supernatants of Artkrohacter P-35 showed (Table I) that the yield of chitinase produced on colloidal chitin was over twice that of any non-chitinous substrate. The monomer of chitin, ~~-acetyl-glucosamine, failed to produce enzyme at both concentrations tested. Glucosamine HCl yielded some enzyme. Growing the organism on disaccharides (lactose and cellobiose) containing glycosidic linkages similar to chitin (beta-1,4) resulted in no enzyme production. Substantial chitinase production occurred when the organism was grown on yeast extract or peptone. Since the exact nature of these substances has not been chemically defined, interpretation of this data is difficult. However, because “yeast extract” is a water soluble extract of dried yeast cells. the possibility exists that soluble oligomers of yeast wall fractions could be present and that these induce chitinase production. Although some acidic metabolites were produced (Table I). the pH of the media did not fall below 5.65. well within the growth range tolerated by the bacterium. Unknown impurities in reagents used in the preparation of the media may have stimulated enzyme formation. Mandels et al. (1962) found that sophorose present in reagent grade glucose was a powerful inducer of cellulase in Trickodermu viride. When Artkr~~~~acterP-35 was grown on colloidal chitin, chitinase was liberated during the exponential phase (Fig. I), suggesting that the enzyme is extracellular. Grinding chitin flakes in a Wiley mill or adding Tween 80 to the culture media substantially increased chitinase production (Table 2). Production of enzyme on 16 mesh milled chitin was greater Table I. EfFect of various carbon sources on chitinasc production after 72 h incubation
Chitinase
l-.
NO. OF P35
0-o
PROTEIN
&--A
CHITINASE
production
by a fusarolytic
Arthrobacter
25
CELLS
8-
6)r- -
TIME
Fig.
Table
(h
1
1. Liberation of chitinase during growth on colloidal
DAYS)
by Arthrobacter chitin (4 mg/ml).
2. Effect of modification of chitin production after 94 h incubation
P-3.5
Fig. 2. Accumulation milled chitin
mesh). The
medium contained 0.05% salts solution.
increased
until Unlike colloidal completely digested
on chitinase
was not experiment.
at the
end
chitin of the
chitin did not even incubation. from to increased from 225 to 300 milliunits, while caused a doubling enzyme concentration. chitin was raised 2.0%, however, enzyme production occurred. The relationship enzyme production. 2 chitin enzyme
than that of colloidal chitin (without Tween SO). Greater accessibility to enzymatic attack (and therefore greater enzyme production) probably resulted from increasing the surface area of the flakes by milling. A similar stimulatory effect was obtained by the addition of Tween 80. Large increases were observed in the case of chitin flakes (4 fold) and F~~auilrrn mycelium (over 3 fold). N-deacetylation of chitin to form chitosan was sufficient to prevent enzyme production. Monreal and Reese (1969) also reported the inability of chitosan to induce chitinase in a strain of Serratia marcescens. The decrease in chitinase production on 24 mesh/cm chitin is attributed to the tendency of the finer particles to adhere to the walls of the culture flasks. Addjtion of Tween 80 overcame this problem and resulted in the highest chitinase production. The formation of chitinase resulting from growth on F. roseurn mycelium indicates that the fungus contains substances necessary for the production of the chitin in hyphal walls 1965). The high of enzyme chitin compared other carbon (Table 1) and the fact that modification chitin to chitosan prevents chitinase (Table 2) indicates enzyme is inducible.
chitin stable in culture.
enzyme
milled was extremely
during 7 days of incubation.
After
chitin
is
presented milled
chitin
caused
an
increase When
slight increase the concentration increase
(Fig. 3a). 2.0%, a enzyme
was
greatly
de-
pressed. chitin was employed growth
substrate
(Fig.
products.
Other
uptake hydrolytic
chitin
that when readily added to cultures, repression occurs. Before enzyme prothese readily utilizable first be consumed (Mandels and Reese, 1960; and Parrish, 1969; Monreal Reese, 1969). these experiments, enzyme production probably steady condition. enzyme production promotes chitin hydrolysis until a is reached where enzyme synthesis. point, the maximum concentration exist, The concentration chitin therefore effect on the rate of hydrolysate liberation. Table shows the effect adding X0 (sorbitan polyoxyethylene monooleate) yield of chitinase. Statistically significant
utilizable catabolic duction
have
of the
reports
w1
26
R. F. MORRISSEY et al.
0.6 2.4 I.2 3.0 1.8
200 *50 100 130 50
n
-b
n Llnll 0
20
0.45
MILLED
CHITIN
0
(%I
0.623
COLLOIDAL
1.25
CHITIN
(mg f ml
)
Fig. 3. Maximum amounts of chitinase and rz-acetyl-glucosamine liberated in cultures various concentrations of (a) milled chitin (I6 mesh) after 120 h incubation and (h) colloidal 70 h incubation. Table
3. Effect of the addition of Tween 80 on chitinase production after 120 h incubation
increases (P
b
80 t 60
40
m
O’ 0
F-H
Fig. 4. Effect
I ’
s mL
40 TEMPERATIJRE 20
of (a) pH and (b) temperature activity.
60 c“C,
0
’
80
on chitinase
containing chitin after
mately 25% of the maximum activity remained at pH 8.5 in 50 mM Tris-HCI buffer. The optimum temperature for the majority of chitinases has been reported to be 40°C (Jeuniaux, 1963). Monreal and Reese (1969) however, reported an optimum temperature of 50°C for the hydrolysis of colloidal chitin by S. marcescens. The data (Fig. 4b) show an optimum temperature at 50°C. with activity rapidly decreasing beyond this point. About 88% of the enzyme was inactivated after incubation for 60 min at 70°C. Routine enzyme assays were conducted at 37°C. The assignment of unit enzyme activity is based upon a linear relationship between the amount of nacetyl-glucosamine released by the hydrolysis and the concentration of the enzyme. The straight line relationship shown in Fig. 5a represents typical results found during this investigation. The proportionality indicates that the assay was free of inhibitory substances which could cause a decline in activity as the concentration of enzyme is increased. In each experiment requiring the assignment of enzyme units, a portion of the enzyme was tested to assure that the production of n-acetylglucosamine was linear with enzyme concentration over the range for unit activity. F&I-e 5b shows the effect of varying the concentration of colloidal chitin on the liberation of n-acetyl-glucosamine. The graph indicates that approximately 1.6-2.0 tig/ml of colloidal chitin are necessary to saturate the enzyme. This experiment was conducted using an undiluted sample of culture supernatant of high activity (0.380 PM n-acetylglucosamine liberated by I4 ml under standard conditions). Since standard enzyme assays used diluted samples, it follows that the 2.0 mg/ml concentration of colloidal chitin used in standard assays would always be sufficient to saturate. Purification of chitinase Several workers have used (NH&SO, precipitation of chitinases as an important part of their purification scheme. Berger and Reynolds (1958) using the culture supernatant from S. griseus obtained the greatest amount of chitinase with a salt saturation of 70% and a pH of 7.0. Skujins, Pukite and McLaren (1970), also working with 5’. griseus chitinase. used the same conditions appar-
$-
Chitinase production by a fusarolytic A~~lr~b~(~fer
27
loo-
0 0
60
/-
60 0
40-
/
/
-
RELATIVEDILUTION OFCULTURE SUPERNATANT
COLLOIDAL CHITIN (mg/mll
Fig. 5. Effect of (a) enzyme concentration and (b) substrate concentration n-acetyl-glucosamine
ently with good results. However, for the purification of Streptomyces antibioticus chitinase, Jeuniaux (1963) collected a 30-50% saturated fraction at a pH of 5-2. This followed adsorption and desorption from chitin. Monreal and Reese (1969) obtained high yields with a 40-80% fraction for the chitinase of S. marcescens. In our work the precipitate obtained at 30% saturation had relatively low specific activity and was discarded. The 30-90% fraction resulted in a 3 fold increase in specific activity. However, one-third of the total chitinase activity was lost with this procedure (Table 4). Skujins et a/. (1970) reported that at pH 8.9 S. griseus chitinase passed through a DEAF-cellulose column while other proteins and phenolic compounds remained in the column. However, with our Art~r~~acter chitinase, much of the enzyme became bound to the column resulting in a two-thirds loss in enzyme units and very little increase in specific activity. These results agree somewhat with the S. marcescens chitinase of Monreal and Reese (1969) which could not be recovered in good quantities from a DEAESephadex column. Skujins et al. (1970) used hydroxypapatite column chromatography to remove proteinase activity from their chitinase preparation. Several attempts to use this technique demonstrated that very little Arthrohacter chitinase could be eluted from the column and the use of hydroxyapatite was abandoned. Chitinase was separated from many other proteins on G-75 Sephadex. The effectiveness of the gel chromatography is illustrated by the elution profile (Fig. 6). Through this procedure a 3 fold increase in specific activity was achieved (Table 4). Table 4. Purification of chitinase
on the production
of
from colloidal chitin.
Fig. 6. Elution profile of chitinase from a G-75 Sephadex column. Fractions 20 through 27 were pooled. The final preparation
resulted
in specific supernatant.
compared
activity
in a 12 foId increase
with the culture
Acknowledgements-Scientific Contribution No. 608, Storrs Agricultural Experiment Station, The University of Connecticut, Storrs, Connecticut 06268, U.S.A. REFERENCES ARONSON J. M. (196.5)The celi wall. In The Fungi, Vol. I,
(G. C. Ainsworth and A. S. Sussman, Eds) pp. 49-76. Academic Press, New York. BERCERL. R. and REYNOLDSD. M. (1958) The chitinase system of a strain of ~treptomyces griseus. Riochem. hiophys. Acta 29, 522-534. JEUNIAUXC. (1963) Chitine et Chitinolyse. Masson et tie, Paris. KOTHSJ. S. and GUNNERH. B. (1967) Establishment of a rhizosphere microflora on carnation as a means of plant protection in steamed greenhouse soils. Proc. Am. Sot. hod. Sci. 91, 617-626. LOWRY 0. H., R~SEBROUGHN. J., FARR A. L. and RANDALLR. J. (1951) Protein measurement with the folin phenol reagnet. J. biol. Chem. 193, 265-275. LINCAPPAY. and LOCKWOOD J. L. (1962) Chitin media for selective isolation and culture of actinomycetes. Phyfopafho~o~y
52, 3 17-323.
MANDELS M., PARRISH F. W. and REESE E. T. (1962) Sophorose as an inducer of cellulase in Trichoderma viride. .I. Bact. 83, 400-408.
MANDELS M. and REESE E. T. (1960) Induction of cellulase in fungi by cellobiose. J. Bact. 79, 816-826. MONREALJ. and REESE E. T. (1969) The chitinase of Serratia marcescens. Can. J. Microbial. 15,689-696. POLLOCKM. R. (1962) Exoenzymes. In The Bacteria, Vol. IV (I. C. Gunsalus and R. Y. Stainer, Eds) 121-178. Academic Press, New York.
28
R. F. M~RRISSEY et al.
REESE E. T.. LOLA J. E. and PARRISH F. W. (1969) Modified substrates and modified products as inducers of carbohydrases. J. Bact. 100, 1151-1154. REISSIG J. R., STROMINGERJ. L. and LELOIR (1955) A modified calorimetric method for the estimation of ,I-acetylamino sugars. J. hiol. Chem. 217, 959-966.
REYNOLDS D. M. (1954) Exocellular chitinase from a streptomycete sp. J. gen. Microbial. 11, IX&159. SKUJINS J., PUKITE A. and MCLAREN A. D. (1970) Chitinase of Streptomyces sp.: purification and properties. Enzymologia 39, 353-370.