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Optimization of Cultural Conditions for the Production of Antifungal Chitinase

by Streptomyces Sporovirgulis

Article in Applied Biochemistry and Microbiology · March 2013

DOI: 10.1134/S0003683813020014

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ISSN 00036838, Applied Biochemistry and Microbiology, 2013, Vol. 49, No. 2, pp. 154–159. © Pleiades Publishing, Inc., 2013.

Optimization of Cultural Conditions for the Production

of Antifungal Chitinase by Streptomyces Sporovirgulis1
M. Swiontek Brzezinskaa, U. Jankiewiczb and K. Lisieckia
a
Department of Environmental Microbiology and Biotechnology, Faculty of Biology and Environment Protection,
Nicolaus Copernicus University, Torun, 87–100 Poland
b Department of Biochemistry, Warsaw University of Life Sciences, SGGW, Warsaw, 02–776 Poland

email: [email protected]
Received April 20, 2012

Abstract—Actinomycetes were screened from soil in the centre of Poland on chitin medium. Amongst 30 iso
lated strains one with high activity of chitinase was selected. It was identified as Streptomyces sporovirgulis.
Chitinase activity was detected from the second day of cultivation, then increased gradually and reached max
imum after 4 days. The maximum chitinase production was observed at pH 8.0 and 25–30°C in the medium
with sodium caseinate and asparagine as carbon and nitrogen sources and with shrimp shell waste as inducer
of enzyme. Chitinase of S. sporovirgulis was purified from a culture medium by fractionation with ammonium
sulphate as well as by chitin affinity chromatography. The molecular weight of the enzyme was 27 kDa. The
optimum temperature and pH for the chitinase were 40°C and pH 8.0. The enzyme activity was characterised
by high stability at the temperatures between 35 and 40°C after 240 min of preincubation. The activity of the
enzyme was strongly inhibited in the presence of Pb2+, Hg2+ and stabilized by the ions Mg2+. Purified chiti
nase from S. sporovirgulis inhibited growth of fungal phytopathogen Alternaria alternata. Additionally, the
crude chitinase inhibited the growth of potential phytopathogens such as Penicillium purpurogenum and Pen
illium sp.
DOI: 10.1134/S0003683813020014

1
Chitinases belong to glycoside hydrolases which types of plant pathogens. Normally, chemical fungi
catalyze the process of chitin degradation. They are cides are used to eliminate their spread. Nevertheless,
produced by microorganisms, insects, plants and ani over the last 40 years these compounds have been used
mals and can be found in a human serum [1]. Various excessively which resulted in pollution and environ
organisms produce different chitinolytic enzymes mental degradation. Their application might lead to
having different characteristics connected with physi death of beneficial insects and microorganisms in the
ological processes. As for bacteria, chitinases play an soil. What is more, they can infiltrate the food chain
important role in nourishment, parasitism and chitin [6]. Currently, certain pesticides are being searched in
recycling. In fungi, protozoa and invertebrates, chiti order to replace the fungicides. Microorganisms
nases are involved in the process of morphogenesis. which produce substances inhibiting the development
When it comes to land plants, vertebrates and humans, of phytopathogenic fungi have increasing popularity.
chitinases constituted defensive mechanisms against Such fungicides are nontoxic or harmful to the
pathogens [1–3]. Chitinolytic enzymes are widely humans and environment. For these reasons, they
used, for instance in preparation of crucial chitooli might be an excellent alternative for chemical fungi
gosaccharides and N–acetyl–D–glucosamines, uni cides.
cellular protein or in the process of protoplasts isola The aim of this research was to isolate chitinolytic
tion from fungi. Moreover, they are employed in actinomycetes and purify chitinase from the micro
examining pathogenic fungi, chitin waste treatment bial strain with the highest enzyme activity. Then, the
and controlling of dissemination of malaria [4]. Pro S. sporovirgulis chitinase was tested against different
duction of inexpensive chitinolytic enzymes is signifi fungal phytopathogens.
cant in chitin waste recycling as it is beneficial for the
environment protection. Predominantly however,
they can be used as the means of biological control [2]. MATERIALS AND METHODS
Plant diseases are one of the major obstacles in cul Screening and Isolation of Actinomycetes. Soil
tivation as they cause a 10% loss in a global agricultural samples from Torun, Przysiek and Che l mza (Poland)
production [5]. Fungi belong to the most aggressive were taken in order to isolate chitinolytic actino
mycetes. Isolation of actinomycetes was done on solid
1 The article is published in the original.
medium Difco Actinomycetes Agar (Merck, United

154
 OPTIMIZATION OF CULTURAL CONDITIONS FOR THE PRODUCTION 155

States) with addition of colloidal chitin (150 g/L). enzyme solution and incubated for 2 h at 4°C. The
Colloidal chitin was prepared according to Lingappa mixture was centrifuged (10.000 × g, 15 min) and the
and Lockwood [7]. The incubation period lasted for supernatant was discarded. The enzyme protein was
7 days at the temperature of 28°C. During the process eluted by 50 mM acetate buffer (pH 4.0) for 30 min.
of incubation the appearing of halo around the colony The obtained preparations were centrifuged and dia
proving that actinomycetes may dissolve chitin was lyzed against the same buffer overnight.
also tested. Two colonies with large halos were selected Chitinase Activity. The activity of chitinase was
for further research. determined by using fluorogenic substrate derived
Identification of Slected Actinomycete Strain. from 4methylumbelliferone (MU). The reaction
Among 30 isolated actinomycete strains there was mixture contained: 1 mL of purified chitinase, 0.125 mL
selected one characterized with high activity of chiti of substrate 4methylumbelliferyl N–acetyl–β–D–
nase. It was identified on the basis of biochemical tests glucosaminide solution (the final concentration in a
[8] as well as on the analysis of the sequence of genes sample was 50 μmol/L) and 0.125 mL of 50 mM phos
encoding 16S rRNA. Amplification of the gene 16S phate buffer (pH 7.0). The control sample, prior to
rRNA was carried out using the universal primers 27F addition of the substrate, was treated with 0.1 mL solu
and 1401R [9]. The matrix in the reaction was consti tion of HgCl2 (final concentration: 4 mM) in order to
tuted by the genomic DNA separated from cells of deactivate the enzyme. The mixture was incubated in
bacteria in the subsequent stage of the logarithmic the dark for 1 h at 40°C. The enzyme reaction was
growth by applying the method described by Kutchma stopped by adding HgCl2. The released MU was mea
et al [10]. After purification, the product of PCR was sured fluorimetrically at 318 nm excitation and
sequenced in the DNA sequencing and oligonucle 445 nm emission using the Hitachi F 2500 spectroflu
otides synthesis laboratory at Institute of Biochemistry orometer (Japan). One unit of chitinase activity (U)
and Biophysics of Polish Academy of Sciences. The was defined as nmol MU/mL.
obtained nucleotide sequences were compared with Determination of Protein Content. The protein
sequences deposited in available databases of Gen concentration was measured by the Bradford method
Bank/EMBL/DDBJ using the software BLAST. [12] with bovine serum albumin as a standard.
Optimization of Culture Conditions. Cultivation of Characterization of Purified Chitinase. The molec
actinomycetes was performed on the media containing ular mass of the chitinase was determined by SDS–
different sources of carbon and nitrogen: Medium 1 PAGE using 12% gel, according to method of Laem
contained (g/L): sodium caseinate—2; asparagine— mli [13] in Trisglycine buffer (pH 8.3). The protein
0.1; K2HPO4—0.5; MgSO4 ⋅ 7H2O—0.1; FeSO4 ⋅ bands were visualized using Coomassie brilliant blue
7H2O—0.01. Medium 2 consisted of (g/L): starch— R250. The molecular weight standards were used, i.e.
10; casein—0.3; KNO3—2.0; NaCl—2.0; phosphorylase b (97 kDa), bovine serum albumin
K2HPO4—2.0; MgSO4 ⋅ 7H2O—0.01; CaCO3— (66 kDa), ovalbumin (45 kDa), carbonic anhydrase
0.02; FeSO4 ⋅ 7H2O—0.01. Medium 3 contained (31 kDa), soybean trypsin inhibitor (21 kDa) and
(g/L): K2HPO4—0.7; KH2PO4—0.3; MgSO4 ⋅ lysozyme (14 kDa). The optimum temperature and
7H2O—0.5; ZnSO4—0.001. The pH of all media was pH for the S. sporovirgulis chitinase were determined.
adjusted to 7.5. The reaction was carried out at various temperatures
Cultivation was performed at the temperatures ranging from 20 to 60°C and pH 4.0–8.0. The thermal
from 20 to 35°C for 6 days with shaking (100 rpm). The stability was also determined. The enzyme was initially
substrates used to produce chitinases were shrimp shell preincubated at different temperatures (30, 40, 50
waste, crab shell chitin and colloidal chitin at 2% and 60°C) for a range of time intervals (0, 60, 120, 180
(w/v) concentration. and 240 min). Afterwards the heat treatment samples
Chitinase Purification. To purify the S. sporovirgulis were cooled and assayed for residual activity at the
chitinase 2 stages were applied: fractionation with temperature of 40°C. The enzyme was preincubated
ammonium sulphate and chitin affinity chromatogra with different metals (Mg, Ca, Pb, Hg, Zn). Final
phy. All purification procedures were carried out at concentration of metals was 1mM. After 30 min, the
4°C. Cultivation was performed at 25°C for 6 days on remaining chitinase activity was measured using the
medium 1 with 2% shrimp shell waste used as the standard assay.
chitinase inductor. After centrifugation of the culture Antifungal activity of crude and purified actino
at 10.000 × g for 20 min, 2l supernatant was precipi mycete chitinase was also determined according to the
tated with ammonium sulphate to 85% saturation. The modified method of Roberts and Selitrennikoff [14].
precipitate obtained by centrifugation (16.000 × g, The chitinase was tested for inhibitory activity against
30 min) was dissolved in 50 mM sodium phosphate growth of phytopathogen fungi: Alternaria alternata
buffer (pH 7.0) and immediately dialyzed against the (isolated from kohlrabi), Fusarium oxysporum (iso
same buffer overnight. The second step of purification lated from potato) Fusarium solani (isolated from
was chitin affinity chromatography made according to parsley) and Botrytis cinerea (isolated from tomato).
the modified method of Escott et al. [11]. Colloidal The fungal strains were obtained from the Bank of
chitin (1 % w/v) was added to the same volume of Plant Pathogens and Studies on their Diversity

APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 49 No. 2 2013

156 SWIONTEK BRZEZINSKA et al.

Table 1. Isolation of chitinolytic actinomycetes from soil shell waste and crab shell chitin powder (Fig. 2). The
samples taken from different places of Poland chitinase activity was revealed after 2 days of incuba
tion with media containing different enzyme inducers,
Number
Sampling pH Number
of chitinolytic
whereas the optimal activity was observed after 4 days
location of soil of soil samples of incubation with shrimp shell waste. The maximum
actinomycetes isolated
chitinase production was observed at pH 8.5 and 25–
Przysiek 6.8–7.2 12 14 30°C (Fig. 3).
Chelmza 7.4–8.5 10 11 The basic factors influencing chitinases production
Torun 5.5–6.2 13 5 are: appropriate medium, type of chitin substance,
temperature and period of cultivation. Conditions in
which the cultivation is performed are individual for
(Poznan, Poland). Besides that, fungi affecting garden each microbial strain producing the enzyme. In this
plants were tested with actinomycete chitinase. For report, the chitinase production by S. sporovirgulis was
their isolation, the leaf blades of cucumber, grass pea, first optimized. Mane and Deshmukh [19] investi
potato and tomato having pathological symptoms, gated chitin degradation caused by three types of acti
were used. Then identification of fungal species was nomycetes: Streptomyces canus, Streptomyces pseudo
determined on the basis of the following literature ref griseolus and Micromonospora brevicatiana. Chitinases
erences: Barnett and Barry [15], Fasatiova [16], production was performed at 35°C. S. canus and
Kwasna et al. [17], and Piontek [18]. S. pseudogriseolus produced chitinase at different level
after 84 h of cultivation whereas M. brevicatiana pro
duced the enzyme after 96 h. There was a gradual
RESULTS AND DISCUSSION increase in chitinase production up to 144 h after
which chitinase activity decreased. The starting pH of
Screening and Isolation of Actinomycetes. 30 strains production medium was 8.0, it declined to 6.5 within
of actinomycetes were isolated from the soil samples the first 60 h of cultivation after which it increased up
taken from different places of Poland (Table 1). These to 8.5 during 144 h. Margino et al. [20] observed chiti
strains were characterized with different chitinolytic nase production on the optimal medium for Strepto
activities fluctuating from 0.33 U/mL to 5.52 U/mL. myces sp. IK which was previously isolated from com
The highest activity was identified in the strain P9 post. This strain was producing chitinases on medium
selected for further research. containing 0.2% of colloid chitin at pH 7.0 and 30°C
Identification of Selected Actinomycete Strain. The after 96 h. Shanmugaiah et al. [21] isolated chiti
procedure of identification of the actinomycete strain nolytic bacteria of Bacillus type from soil and rhizo
P9 was performed as described in methods. The sphere. They also optimized the cultivation conditions
selcted microbial strain was identified as Streptomyces of Bacillus laterosporous having the highest activity.
sporovirgulis (accession number AB704795) The maximum chitinase production was observed in
Optimization of Culture Conditions. The best yeast nitrogen based medium amended with 0.3% col
medium for Streptomyces sporovirgulis chitinases pro loidal chitin at pH 8.0 and 35°C after 4 days of inocu
duction was medium 1 containing asparagine and lation.
sodium caseinate as the sources of carbon and nitro Actinomycetes produce chitinases constitutively,
gen (Fig. 1). Among the substrates essential to produce however the appropriate chitin substrate increases
chitinases the most effective inductors were shrimp their production. Using chitin substance to chitinases

U/mL U/mL
3.0 5
2.5 4 3
2.0 3
2
1.5
2
1.0 1
1
0.5
0 0
Medium 1 Medium 2 Medium 3 2 4 6 8 10
Cultivation, days

Fig. 1. Effect of composition medium on production of Fig. 2. Effect of chitinous substances on production of
chitinase from S. sporovirgulis. Cultivation was performed chitinase from S. sporovirgulis. Cultivation was performed
at 25°C and pH 8.5 with 2% shrimp shell waste as enzyme at 25°C on medium 1 at pH 8.5. 1—colloidal chitin; 2—
inducer. Vertical bars represent standard deviation (n = 3). shrimp shell waste; 3—crab shell chitin.

APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 49 No. 2 2013

 OPTIMIZATION OF CULTURAL CONDITIONS FOR THE PRODUCTION 157

production depends individually on the microbial U/mL

strain producing the enzyme. In numerous experi 9 (a)
ments colloidal chitin is used as a substrate to produce 8
chitinases [19, 22]. There also exist microorganisms 7
which produce chitinases in the presence of shrimp 6
shells [23]. Rattanakit et al. [24] reported that shrimp 5
waste is the appropriate substrate to cultivate Aspergil 4
lus sp. S1–13 on solid medium. This organism synthe 3
sizes the same or even greater amount of enzymes than 2
fungi cultivated on the medium containing colloidal 1
chitin. 0
5.5 7.0 8.5
Chitinase Purification. To purify chitinase from pH
S. sporovirgulis precipitation with ammonium sulfate 6 (b)
and chitin affinity chromatography were used. This 5
procedure resulted in 2.3–fold purified enzyme prep 4
aration with a specific activity of 24.8 U/mg and 12 % 3
recovery of protein (Table 2). There are several tech 2
niques which might be applied to purify chitinases. 1
Chitin affinity chromatography seems to be the best 0
choice. Nevertheless, researchers used various meth 20 25 30 35
ods. To purify chitinases Streptomyces venezuelae P10, °C
Mukherjee and Sen [25] applied precipitation with
ammonium sulfate (80%) and affinity binding to
Fig. 3. Effect of medium pH (a) and temperature (b) on
chitin. Additionally, they implied the third stage of production of chitinase from S. sporovirgulis. Cultivation
purification—DEAEcellulose column. The active was performed at 25°C (a) and pH 8.5 (b) on medium 1
fractions were pooled, resulting in 3.2fold total puri with 2% shrimp shell waste. Vertical bars represent stan
fication with a specific activity of 26.7 U/mg and 2.1% dard deviation (n = 3).
recovery of protein. Nawani and Kapadnis [26]
reported 5.1fold purification of S. marcescens NK1
chitinase with ammonium sulfate precipitation fol Ca2+ and Mg2+ ions stabilazed purified chitinase
lowed by Sephadex G–100 gel filtration. Aly et al. [27] whereas Hg2+ and Pb2+ caused the complete inhibition of
purified chitinase Streptomyces anulatus using ammo the enzyme activity. Chitinase of S. sporovirgulis revealed
nium sulfate precipitation, DEAE–cellulose ion antifungal features in relation to certain types of phyto
exchange chromatography and Sephadex G–100 gel pathogens. Purified actinomycete chitinase inhibited
filtration. Jiang et al. [28] after numerous stages of growth of fungal phytopathogen Alternaria alternata.
purification obtained enzyme from Streptomyces rose Additionally, the crude chitinase from S. sporovirgulis
lus with relatively high recovery of activity of 34% and inhibited the growth of potential phytopathogens Penicil
specific activity of 30 U/mg. lium purpurogenum and Penillium sp. (Table 3).
Characteristic of Chitinase. Electrophoresis of The bacterial chitinases were reported to be in the
purified chitinase from S. sporovirgulis in denaturing range of 20–60 kDa [29]. The molecular weight of the
conditions showed presence of a single band of protein purified chitinase from Streptomyces anulatus was
with the molecular weight of about 27 kDa (Fig. 4). determined about 28 kDa [27]. Jiang et al. [28]
The enzyme was thermally stable between 30–40°C reported that molecular mass of chitinase Streptomyces
and after 240 min preincubation only a slight decrease roseolus was about 40 kDa. Chitinases produced by
in its activity was observed (Fig. 5). After a 120 min this strain of actinomycetes demonstrated the maxi
preincubation at 50°C, chitinase kept only as much as mum activity at 60°C and pH 6.0. Their activity was
20% of its activity. Preincubation of the enzyme at the stabilized by Mg2+, Ba2+ and Ca2+ ions and inhibited
temperature of 60°C led to entire disappearance of its to different extent by Cu2+, Co2+, Mn2+ ions. Chiti
activity. Optimal temperature and pH for the S. sporovir nase from Streptomyces griseus was optimally active at
gulis chitinase activity were 40°C and pH 8.0. pH 6.0 and 40°C. The enzyme was stable from pH 5.0

Table 2. Purification of extracellular chitinase from S. sporovirgulis

Step Total protein, mg Total activity, U Specific activity, U/mg Yield, % Purification factor
Culture supernatant 300 3200 10.7 100 1
NH4SO4 (85%) 75 1125 15.0 35 1.4
Chitin affinity chromatography 15 372 24.8 12 2.3

APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 49 No. 2 2013

158 SWIONTEK BRZEZINSKA et al.

kDa M 1 Relative activity, %

100 1
66 90
80 2
70
45 60
50
40
30
35 20
10 4 3
25 0 60 120 180 240
min
18
Fig. 5. Thermal stability of purified chitinase S. sporovirgu
14 lis. 1—30°C; 2—40°C; 3—50°C; 4—60°C.

Fig. 4. SDS–PAGE analysis of purified chitinases from

S. sporovirgulis. M—molecular weight markers; 1—puri Rhizoctonia bataticola [33]. The purified chitinase
fied chitinase preparation. from Streptomyces sp. M–20 showed antifungal activ
ity against Botrytis cinerea [31]. Chitinase from Strep
tomyces plicatus inhibited growth Rhizoctonia solani,
to 9.0 and between 20–50°C. The molecular mass of Sclerotinia sclerotiorum, Fusarium graminearum,
the purified chitinase was 34 kDa [30]. Chitinase Strepto Fusarium solani, Rosellinia necatrix and Pythium aph
myces sp. M–20 was optimally active at pH 5.0 and 30°C. anidermatum [34].
The enzyme was stable from pH 4.0 to 8.0, and up to Present study is the first case demonstrating inhib
40°C. Among the metals and inhibitors that were tested, iting activity of actinomycete S. sporovirgulis against
the Hg+, Hg2+ and pchloromercuribenzoic acid com fungi Alternaria alternata, Penicillium purpurogenum
pletely inhibited the enzyme activity [31]. and Penillium sp.
Bacterial chitinases have great antifungal potential.
Research done by Bhushan and Hoondal [32] showed ACKNOWLEDGMENTS
that chitinase from Bacillus sp. BG—11 was notably
compatible with commonly used fungicides and insec The research was financed by the National Science
ticides. What is more, they revealed significant activity Center (Poland), with grant no. N N304 373538.
against fungal phytopathogens. Chitinases produced
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