1224 Chiang Mai J. Sci.

2017; 44(4)

Chiang Mai J. Sci. 2017; 44(4) : 1215-1221

http://epg.science.cmu.ac.th/ejournal/
Contributed Paper

Production of Chitooligosaccharides with

Antibacterial Potential via Crude Chitinase Enzymes
from Marine Fungi
Apiradee Pilantanapak* [a], Yaowapha Waiprib [b], Phattharawadee Eadtem [b] and
Watanalai Panbangred [c,d]
[a] Department of Microbiology, Faculty of Science, Burapha University, Chonburi 20131, Thailand.
[b] Department of Food Technology, Faculty of Science, Burapha University, Chonburi 20131, Thailand;
current address: Department of Fishery Products, Faculty of Fisheries, Kasetsart University,
Bangkok 10900.
[c] Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
[d] Mahidol University-Osaka University Collaborative Research Center on Bioscience and Biotechnology
(MU-OU CRC), Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
* Author for correspondence; e-mail: [email protected]
Received: 8 January 2017
Accepted: 9 March 2017

ABSTRACT
Twenty marine fungi from driftwoods and Nypa fruticans in the marine environments
were screened for chitinase production in culture filtrates. Two best strains with the highest
chitinase activity were selected to produce crude chitinase enzyme. Thereafter chitooligosaccharide
mixtures were produced by hydrolyzing native chitosan with crude chitinase fungal enzyme
and these were then assess for their antibacterial activity against 2 Gram positive and 2 Gram
negative bacteria. Two marine fungi Astrosphaeriella sp. BUSK 55-1 and Oxydothis sp. BUSK
43-2, were selected for this study. The chitinase activities at day 21 were 16.9 mU/mL and
22 mU/mL, respectively. Hydrolysis of native chitosan (720 kDa, 86.5 % degree of
deacetylation) by approximate 0.8U crude chitinase of these two marine fungi yielded
chitooligosaccharides mixtures with an average molecular weight of 8.54 kDa-8.62 kDa.
Using the standard disc paper method, the chitooligsaccharides displayed antibacterial activity
against all tested bacteria; Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus
subtilis. The best activity was oligosaccharides prepared from chitinase enzyme of Astrosphaeriella
sp. BUSK 55-1. The standard disc contained at least 250 ppm and at least 500 ppm
oligosaccharides obtained from this enzyme displayed inhibition against B. subtilis and S. aureus,
respectively.

Keywords: chitooligosaccharides, antibacterial activity, marine fungi

1. INTRODUCTION
In nature, chitin and chitosan are - D-glucosamine) is a water insoluble polymer
associated polymers with variable degrees of that is found abundantly in the exoskeletons
deacetylation. Chitin (poly - β-1→4 N-acetyl of crustaceans and insects [1, 2]. Chitin can
Chiang Mai J. Sci. 2017; 44(4) 1225

be hydrolyzed by chitinase to produce driftwood or from the mangrove plant

different amounts of chitooligosaccharides Nypa fruticans growing in the intertidal estuarine
and N-acetyl oligosaccharide mixtures regions of Chonburi and SamutSongkram
depending on the degree of deacetylation Province, Thailand. Samples of the axenic
(DD) [3-5]. Their specificity and safety of cultures were then kept as stock cultures
chitooligosaccharides and the derived in the Department of Microbiology, Faculty
N-acetyl oligosaccharide has led to their of Science, Burapha University.
broad study and use within medicine and The bacterial strains E. coli ATCC 25922
industry, most notably as antimicrobial and S. aureus ATCC 25923 together with
substances and as an enhancer of immunity environmental isolates of Bacillus subtilis
[2, 6-8]. Chitooligosaccharides have wider and Pseudomonas aeruginosa were used as
application than their native forms due to tested bacteria for antibacterial activity of
their solubility properties [9]. An alternative chitooligosaccharides. All bacteria were kept
method to obtain chitooligosaccharides is as stock cultures in the Department of
by acid hydrolysis, however, enzymatic Microbiology, Faculty of Science, Burapha
hydrolysis is a gentler process allowing for University. Identification was confirmed by
easier control of molecular weight [6]. using biochemical methods as detailed
In several reports, the chitooligosaccharides within the Bergey’s manuals for determinative
used as antimicrobial agents were produced bacteriology [20].
from several purified or commercial enzymes Shrimp chitin was prepared from Penaeus
[10-12]. In this case, the generation of large merquiensis, whilst the chitosan, with an
amounts of product is limited by the high 86.5 % degree of deacetylation (86.5% DD),
cost of commercial enzymes [12-14]. Recently, was prepared from the P. merquiensis chitin.
chitooligosaccharides obtained by crude The degree of deacetylation was determined
enzyme hydrolysis using microbial enzymes by acid base titration according to the method
have been employed as an alternative method detailed in Toei and Kohara [21]. The
[14-15]. commercial chitinase used in this study
Marine fungi have been shown to be was derived from Streptomyces griseus and
a rich source of chitinase [8, 16-18]. To the purchased through Sigma, Germany.
authors’ knowledge, however, the production
of chitooligosaccharides using crude 2.2 Preparation of Fungal Cultures
chitinase from marine fungi has been rarely Twenty fungi collected from the marine
reported [19]. This study, therefore, aimed environments were sub-cultured from stock
to evaluate several marine fungi as new cultures and placed on potato dextrose agar,
sources of chitinase for the production prepared with natural seawater (25 ppt).
of chitooligosaccharides and then to One piece of mycelium was cut from each
test the inhibitory effect of the derived active growing culture and was grown in
chitooligosaccharides against selected potato dextrose broth and then screened
bacteria. for enzyme.

2. MATERIALS AND METHODS 2.3 Preparation of Colloid

2.1 Fungal Strains, Bacterial Strains, Colloidal chitin was prepared by
Chitosan and Enzymes dissolving 10g chitin in 100 ml 85%
Marine fungi were isolated from either phosphoric acid and then placing them in
1226 Chiang Mai J. Sci. 2017; 44(4)

an incubator shaker at 180 rpm, 30 °C for Reissig et al. [23] using 1 ml of 0-20 μg/ml
18h. The hydrolysate was washed with 1L N-acetylglucosamine as a standard. One unit
distilled water with continuous stirring for of enzyme activity (1U) was defined as the
10 min. Colloidal chitin was collected amount of enzyme that catalyzes the release
by centrifugation at 10,000 rpm at 4 °C for of 1 μmole of N-acetylglucosamine or its
10 min. The washing process was repeated equivalent per one ml of enzyme in one min.
5-6 times until the pH of the water was
about pH 5.5. The pH of colloid was 2.5 Chitosanase Assay
subsequently adjusted to pH 6 with 1M A 200 μl aliquot of the culture filtrate
NaOH and the colloidal chitin collected was reacted with 200 μ l of 5% colloidal
following centrifugation at 10,000 rpm at chitosan in 50 mM acetate buffer, pH 6.0
4 °C for 10 minutes; the resultant product at 50 °C for 60 min. Here, the concentration
was kept as a 50% stock culture. of the reducing sugar was measured using
Colloidal chitosan was prepared as a modification of the colorimetric DNS
previously described [22] with minor method [24] using 1 ml of 0-10 μ g/ml
modification. Briefly 10 g of chitosan was D-glucosamine as the standard. One unit of
mixed with 100 ml 85% phosphoric and enzyme activity was defined as the amount
was kept at 4 °C for 18 hours. One liter of of enzyme that catalyzes the release of
distilled water was added to the hydrolysate 1 μmole of glucosamine or its equivalent
and the colloid was precipitated by adding per one ml of enzyme in one min.
2 volumes cold ethanol, followed by
centrifugation at 10,000 rpm. The pellet 2.6 Protein Determination
was repeatedly washed with cold ethanol Protein concentration was determined
until the pH was 5.5 and was kept as 50% spectrophotometry at 595 nm by using
stock solution in water. bovine serum albumin as the standard [25].

2.4 Chitinase Assay 2.7 Preparation of Chitooligosaccharides

A 1cm × 1cm piece of agar containing by Fungal Enzymes from Chitosan
mycelia was removed from each fungal The chitosan solution (1% w/v) in
culture and then chopped into smaller pieces 0.05 M acetic acid, pH 4.5 was sterilized by
and then placed into a 250 ml Erlenmeyer autoclaving at 110 °C for 10 min. Seventy
flask containing 50 ml of minimal medium milliliters of the sterile chitosan solution
supplemented with 2% colloidal chitin. was mixed with 70 ml of a 0.05M acetate
The flasks were shaken at 200 rpm for 3, 9, buffer pH 4.5 and 40 ml of crude enzymes
15 21 and 26 days at 25 °C. On the relevant (containing approximately 0.8U of chitinase)
day, 2 ml of culture broth was removed in the fungal culture filtrate. The mixture was
from each culture and then centrifuged at incubated in rotary shaker at 200 rpm/min
8,000 rpm for 10 min to collect the culture at 37 °C and then sampled after one hour
filtrate. An aliquot of 200 μl of the culture and then again at 7 h. After hydrolysis, the
filtrate was then reacted with 200 μl of 5% hydrolysates were boiled for 10 min, chilled
colloidal chitin in 50 mM acetate buffer, on ice and then neutralized with 0.5M
pH 6.0 at 37 °C for 60 min. The reducing sodium hydroxide; all supernatants were
sugar was measured using the method of then filtered before being freeze dried.
Chiang Mai J. Sci. 2017; 44(4) 1227

2.8 Molecular Weight and Yield concentrations of 250, 500, 1,000, 2,000
Determination and 4,000 ppm were applied to sterile
The average molecular weight of the 6 mm diameter paper discs (equivalent to
degraded chitosan was measured by 2.5-40 μg/disc) and then placed on the agar
gel permeation chromatography (GPC, surface. The plates were incubated at 37 °C
PL-GPC110, USA). An Ultraliner Hydrogel for 16-18 h. Positive results were determined
column was used for resolving products by clear zone around the discs. A native
in the range of 1 kDa-20,000 kDa. A mixture chitosan, dissolved in 1% acetic acid (pH 6.0),
of sodium acetate solution (0.5M) and soaked disc was used as the control.
0.5M acetic acid in a ratio of 1:1 was used as
the eluent. A flow rate of 0.6 ml/min was 3. RESULTS
maintained through the column. The resultant 3.1 Characterization of Chitinase
eluents were monitored by RI detector. Producing Strains
The sample concentration were determined Two out of the twenty higher obligate
and diluted to obtain a final concentration of marine fungal strains, Astrosphaeriella sp.
0.2 mg/ml (0.02% w/v). Pullulan (M.W. range BUSK 55-1 from driftwood and Oxydothis
5.8 kDa-1,660 kDa) was used as a standard. sp. BUSK 43-2 from Nypa fruiticans, exhibited
Water soluble chitooligosaccharides the highest chitinase activity in the minimal
were calculated as previously reported [26] broth media supplemented with 2% colloidal
as follows: percent yield is equivalent to chitin. The average chitinase activities of
total weight of water soluble chitosan Astrosphaeriella BUSK 55-1 and Oxydothis sp.
after neutralization divided by total weight BUSK 43-2 were 16.9 mU/ml and 22.0
of chitosan in acetic acid solution and then mU/ml, respectively. Both chitinase positive
multiplied by 100. strains were also analyzed for chitosanase
activity to determine whether only chitinase
2.9 Oligosaccharide Preparation and was present. No chitosanase activity, however,
Screening of Antibacterial Activity was detected.
To prepare a chitooligosaccharides
stock solution, 40 mg chitooligosaccharides 3.2 Molecular Weight Analysis and Yield
was dissolved in 10 ml distilled water of Chitooligosaccharides Produced from
(equivalent to 4,000 part per million (ppm) Chitosan Hydrolysis
and then filtered through a 0.45 μm membrane Before hydrolysis with crude enzymes,
(Millipore Corporation, Bedford, USA). the molecular weight of the native chitosan
The stock solution was twofold diluted to was determined to be about 720 kDa. After
obtain the concentrations ranged from 1 h hydrolysis at 37 °C with approximate
250-2000 ppm. The antibacterial activity of 0.8 U crude fungal chitinase in the culture
the prepared chitooligosaccharides was filtrate, the average molecular weight of the
then determined by the agar disc diffusion chitooligosaccharides prepared from the
method using a modification of the method Astrosphaeriella sp. BUSK 55-1 and Oxydothis
detailed by CLSI (2012). [27]. Approximate sp. BUSK 43-2 enzymes had decreased to
108 cells/ml of indicator bacterial suspensions about 8.62 kDa and 8.54 kDa, respectively
were swabbed using a cotton swab on (Table 2). The ability of chitinase to degrade
Muller Hinton agar plates. Ten microliters of 86.5% DD highlights the non-specific
each chitooligosaccharide solution at chitinase enzyme hydrolysis of chitosan in
1228 Chiang Mai J. Sci. 2017; 44(4)

marine fungi. The non-specific activity was than the yield when using commercial
confirmed by commercial enzyme hydrolysis. enzyme. The percentage yield produced by
The percentage yields of chitooligosaccharides enzyme of Astrosphaeriella sp. BUSK 55-1 and
produced from 0.8U of crude chitinase Oxydothis sp. BUSK 43-2 were not, however,
enzymes were found to be slightly lower markedly different (Table 2).

Table 1. The determined chitinase activities in two marine fungi collected from Thailand.
Both fungi are from Nypa fruticans in Samut Songkram province.
Fungi Average chitinase Average protein Specific
activities (mU/ml) contents (mg/ml) activities (mU/mg)
Astrosphaeriella sp. BUSK 55-1 16.9 1.1 15.4
Oxydothis sp. BUSK 43-2 22 0.8 27.5

Table 2. The percentage yield and average molecular weight of the chitooligosaccharides
obtained from the hydrolysis of chitosan derived from marine chitinase enzyme and commercial
enzymes at 37 °C for 1 h.
Enzyme origin Chitooligosaccharides production
Average MW (kDa) % Yield
Commercial chitinase from S. griseus 7.53 51.7
*Astrosphaeriella sp. BUSK55-1 8.62 40.7
*Oxydothis sp. BUSK43-2 8.54 41.3
* Fungi were cultivated in minimal media containing 2% colloidal chitin.

3.3 Antibacterial Activity bacterial species; B. subtilis and S. aureus

The results provided in Table 3 indicate appear to be inhibited at lower concentrations
that chitooligosaccharides from crude than the Gram negative bacteria; E. coli and
chitinase of the two marine fungi, tested by P. aeruginosa. The chitooligosaccharides at
applied 10 μ l of various concentrations least 250 ppm and at least 500 ppm, obtained
(250-4,000 ppm) to the standard disc, could from the fungal enzyme of Astrosphaeriella sp.
inhibit the growth of several selected bacterial BUSK 55-1 inhibited B. subtilis and S. aureus,
species. The inhibitory activity depended respectively.
on the enzyme sources. The Gram positive

Table 3. Antibacterial activities of the chitooligosaccharides (COS) derived from digestion of

native chitosan with chitinase from BUSK55-1 and BUSK43-2 using disc diffusion susceptibility
test.
COS produced from Minimum inhibitory concentration of COS (ppm)*
E. coli P. aeruginosa S. aureus B. subtilis
Astrosphaeriella sp. BUSK55-1 1000 1000 500 250
Oxydothis sp. BUSK43-2 2000 1000 2000 500
Native chitosan >4000 >4000 >4000 250
*Paper discs soaked with 10 μl of 250, 500, 1000, 2000 and 4000 ppm COS solution which equivalent to 2.5,
5, 10, 20 and 40 μg/disc
Chiang Mai J. Sci. 2017; 44(4) 1229

4. DISCUSSION previously [28, 30]. The inhibition of chito-

The chitinase producing fungi in this study oligosaccharide against Gram negative
are higher obligate marine fungi (sexual bacteria is through the interaction of
stage-ascomycetes). These are in contrast to chitoligosaccharide to cell surface and
most of the previous reports on fungi, where blocking nutrient flow leading to bacterial
the lower fungi and imperfect stages of death, while in Gram positive bacteria the
terrestrial fungi such as Aspergillus, Penicillium interaction of chitoligosaccharide to cell wall
and Cephalosporium have been recorded [16]. disrupts cell wall and cell membrane structure
By comparison, Sherief et al. [17] reported [31].
that the activity of chitinases from seven
terrestrial fungi ranged between 10-30, 5. CONCLUSIONS
820 mU/ml whilst the activity of the two The activities of two crude chitinases
marine chitinases deter mined here, i.e. isolated from two marine fungi were found
16.9 mU /ml for Astrosphaeriella sp. and 22 to degrade native chitosan to produce
mU/ml for Oxydothis sp., come approximately effective chitooligosaccharides. Only 0.8U of
in the middle of the range of species crude chitinase enzymes in culture filtrate can
considered by Sheriff and co-workers [17]. hydrolyze native chitosan of 720 kDa
The level of enzyme activities determined in (86.5%DD) to the appropriate size of ca.
this study, however, are sufficient to 8 kDa in 1 h at 37 °C. The highest percentage
produce active chitooligosaccharides. yield from each source was about 41%.
The results provided in Table 2 show that The chitooligosaccharides derived from
the chitooligosaccharides recovered by the Astrosphaeriella sp. BUSK 55-1 performed the
approaches used in this study have a molecular best in the inhibition study, The standard disc
weight of around 8 kDa. This corresponds contained at least 250 ppm and at least
with the effective molecular weight obtained 500 ppm chitooligosaccharides displayed
from commercial chitinase hydrolysis inhibition against B. subtilis and S. aureus,
(7.5 kDa) and a molecular weight of between respectively. From the findings of this study,
5-10 kDa which has been reported in several it is suggested that marine fungi represent a
published literary accounts [28-29]. good and rich source of chitinase to produce
The percent yields of chitooligosaccharides effective, water soluble chitooligosaccharides
(41.3%) from the crude fungal chitinase in for potential medicinal use in the future.
the culture filtrates were only slightly lower
than those produced from commercial ACKNOWLEDGEMENTS
enzyme from Streptomyces griseus and could This work was partially supported by the
be improved upon by the process of Faculty of Science, Burapha University
optimization. together with the joint research grant between
The chitooligosaccharides produced Thailand Research Fund (TRF) and
from the marine chitinase exhibited better Commission of Higher Education for
antibacterial activity against the Gram positive financial support (CHE) (Grant number
bacterial species than against the Gram MRG5280096). The authors would also like
negative bacteria. This differential inhibition, to thank BIOTEC Culture Collection for
with chitooligosaccharides performing kindly providing the marine fungi used in this
better against Gram positive bacteria than study. We are also grateful to Dr. Surang
Gram negative bacteria, has been reported Thamthiankul for technical assistance with
1230 Chiang Mai J. Sci. 2017; 44(4)

the chitinase assay techniques. We also thank [16] Smitha S.L., Correya N.S. and Philip R.,
Dr. Andrew Shinn for proof reading and Int. J. Res. Mar. Sci., 2014; 3: 5-10.
providing comments on the manuscript. [17] Sherief A.A., El-Sawah M.M.A. and
Abd El-Naby M.A., Appl. Microbiol.
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