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Production of chitosan-oligosaccharides by the chitin-hydrolytic system of Trichoderma harzianum and their antimicrobial and anticancer effects
Journal Pre-proof Production of chitosan-oligosaccharides by the chitin-hydrolytic system of Trichoderma harzianum and their antimicrobial and anticancer effects Olicón-Hernández Dario Rafael, Zepeda-Giraud Luis Fernándo, Pedroza-Torres Abraham, Vázquez-Landaverde Pedro Alberto, Guerra-Sánchez Guadalupe, Pardo Juan Pablo PII:
S0008-6215(19)30494-X
DOI:
https://doi.org/10.1016/j.carres.2019.107836
Reference:
CAR 107836
To appear in:
Carbohydrate Research
Received Date: 22 August 2019 Revised Date:
23 September 2019
Accepted Date: 15 October 2019
Please cite this article as: Olicó.-Herná. Dario Rafael, Z.-G. Luis Fernándo, P.-T. Abraham, Vá.Landaverde. Pedro Alberto, Guerra.-Sá. Guadalupe, P.J. Pablo, Production of chitosan-oligosaccharides by the chitin-hydrolytic system of Trichoderma harzianum and their antimicrobial and anticancer effects, Carbohydrate Research (2019), doi: https://doi.org/10.1016/j.carres.2019.107836. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
1
Production of chitosan-oligosaccharides by the chitin-hydrolytic system of
2
Trichoderma harzianum and their antimicrobial and anticancer effects
3 4
Olicón-Hernández Dario Rafaela; Zepeda-Giraud Luis Fernándob; Pedroza-Torres
5
Abrahamc; Vázquez-Landaverde Pedro Albertod; Guerra-Sánchez Guadalupeb;
6
Pardo Juan Pabloa*.
7 8
a
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de Bioquímica. Laboratorio 7. Circuito Interior s/n, Ciudad Universitaria CP 04510,
Universidad Nacional Autónoma de México. Facultad de Medicina. Departamento
10
Ciudad de México, México.
11
b
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Departamento de Microbiología. Laboratorio de bioquímica y biotecnología de
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hongos. Carpio y Plan de Ayala s/n. Santo Tomas, Miguel Hidalgo. CP 11350,
14
Ciudad de México, México.
15
c
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Hereditario. Avenida San Fernando 22, Belisario Domínguez Secc XVI, CP 14080
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Ciudad de México, México.
18
d
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Tecnología Avanzada, Unidad Querétaro. Cerro Blanco 141. Colinas del
20
Cimatario. CP 76090 Querétaro, México.
Instituto Politécnico Nacional. Escuela Nacional de Ciencias Biológicas.
Cátedra CONACYT-Instituto Nacional de Cancerología. Clínica de Cáncer
Instituto Politécnico Nacional. Centro de Investigación en Ciencia Aplicada y
21 22
*Corresponding author:
23
Pardo Juan Pablo
24
Universidad Nacional Autónoma de México. Facultad de Medicina. Departamento
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de Bioquímica. Laboratorio 7. Circuito Interior s/n, Ciudad Universitaria CP 04510,
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Ciudad de México, México. Phone number: (+52) 555623 2175. Email:
27
[email protected]
28
Abstract
29
Chitosan-oligosaccharides (COS) are low-molecular weight chitosan derivatives
30
with interesting clinical applications. The optimization of both COS production and
31
purification is an important step in the design of an efficient production system and
32
for the exploration of new COS applications. Trichoderma harzianum is an
33
innocuous biocontrol agent that represents a novel biotechnological tool due to the
34
production of extracellular enzymes, including those that produce a COS mixture.
35
In this work, we propose different systems for the production of COS using the T.
36
harzianum chitinolitic system. A complete qualitative and quantitative analysis of a
37
partially purified COS mixture were performed. Also, an evaluation of the
38
anticancer and antimicrobial effects of the COS mixture was carried out. Three
39
chitosan variants (colloidal, solid and solution) and two fungus stages (spores and
40
mycelia) were tested for COS production. The best system consisted of the
41
interaction of the solid chitosan and the fungal spores, producing a COS mixture
42
containing species from the monomer to the hexamer, in a concentration range of
43
7 to 238 mg/mL, according to chromatographic analysis. The proposed purification
44
method isolated the monomer and the dimer from the COS mixture. Moreover, the
45
COS mixture has an inhibitory effect on the growth of bacteria and changes the
46
morphology of yeasts. As anticancer compounds, COS inhibited the growth of
47
cervical cancer cells at concentration of 4 mg/mL and significantly reduced the
48
survival rate of the cells. In conclusion, T. harzianum proved to be an efficient
49
system for COS mixture production.
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Key words: Chitosan-oligosaccharides; Trichoderma harzianum; antimicrobial;
51
anticancer
52 53 54 55 56
57
1. Introduction
58
Trichoderma harzianum is a filamentous ascomycete used as a biocontrol element
59
and biotechnological tool that has gained interest in recent years [1, 2]. This fungus
60
is a cosmopolitan microorganism, with applications in the field of agriculture, being
61
part of commercial mixtures used for composting and involved in the control of
62
pests in crops of interest [3]. The applications of this fungus are not limited to
63
agricultural topics, since it also has the ability to degrade different substrates to
64
produce extracellular enzymes of industrial interest, and has been used for the
65
manufacture of nanoparticles with antibiotic action and the production of biofuels
66
[2, 4-6].
67
It has been reported that T. harzianum has a powerful inhibitory effect against
68
phytopathogenic fungi, with mycoparasitism as one of the most important
69
mechanisms associated with this effect; however, the production of antibiotic
70
molecules as well as the secretion of hydrolytic enzymes that attack the cell wall of
71
different fungi also have an important role in biocontrol behavior, with the group
72
extracellular chitinases being one of the most interesting [7]. In this context, 331
73
enzymes with chitinase activity were reported in the uniprot database for T.
74
harzianum and four of them were identified and characterized as endochitinases
75
(https://www.uniprot.org/) [8]. Possibly, T. harzianum has one of the most versatile
76
"chitinomes" in nature since it is known that some strains have changes in their
77
gene architecture, signal peptide, domain organization and molecular weight in
78
chitinase production [9].
79
Chitinases (EC 3.2.1.14) are endo- and exo- enzymes that degrade chitin, a dense
80
and crystalline N-acetyl-glucosamine-polymer present in insects and crustaceans.
81
In fungi, chitinases contribute to morphogenetic, nutritional and pathogenic
82
processes, including spore germination, hyphal branching, autolysis and
83
mycoparasitic interactions [10]. From a biotechnological point of view, chitinases
84
are used either as bioinsecticides or to obtain bioactive derivatives from chitin
85
and/or chitosan (deacetylated derivative of chitin).
86
Chitosan-oligosaccharides (COS) are low-molecular-weight chains of 6-10
87
glucosamine repeats derived from the action of chitinases on chitosan [11]. COS
88
are potential therapeutic compounds owing to their importance for human health,
89
for example via their proposed use as antibiotics, anticancer and anti-cholesterol
90
molecules [11, 12]. COS are produced by a variety of extracellular enzymes,
91
including chitosanases, chitinases, papain and cellulases [13, 14]; however, many
92
of these only explore production with a single substrate.
93
To our knowledge, there is only one report on the use of chitinase from T.
94
harzianum for the production of COS [15]. In this study, COS mixture was obtained
95
from α and/or β-chitosan and the mixture was effective against bacteria. However,
96
in this work the composition of the COS mixture was not completely identified and
97
the influence of variants of the substrate or the stage within the cell cycle of the
98
fungus were not tested.
99
The goals of this work were as follows: 1) to evaluate six different systems for the
100
production of COS using the chitinase from T. harzianum and the combination of 3
101
variants of chitosan (colloidal, solid and as dilution) and 2 cell cycle stages of the
102
fungus (spores and mycelia); 2) to identify and quantify this COS mixture by
103
chromatographic techniques; 3) to develop a partial purification system and, 4) to
104
search for clinical applications of COS as antimicrobial and anticancer agents.
105
2. Materials and methods
106
2.1.
Strain and culture conditions
107
Trichoderma harzianum strain T1 was isolated from contaminated soil and belongs
108
to a collection of strains in the laboratorio de bioquímica y biotecnología de hongos
109
of ENCB-IPN, México. This strain exhibited tolerance in the biodegradation of
110
petroleum hydrocarbons according to the results reported by Argumedo-Delira et
111
al. [16].
112
Commercial Potato Dextrose Agar and Broth (PDA and PDB, Becton Dickinson
113
Franklin Lakes, NJ, USA) were used for the for the maintenance and storage of the
114
T1 strain.
115
To obtain mycelium, discs of 1 cm diameter were cut from PDA plates with
116
previous growth and placed on fresh plates. Discs were cultured at 28°C under
117
static conditions until the mycelium occupied the entire plate. The collection of
118
spores was obtained by mechanical processes using plates saturated with
119
mycelium, according to our previous protocol and adjusted by the Neubauer
120
chamber method [17].
121
2.2.
Chitinase induction
122
For chitin induction, a production medium was designed based on the method
123
outlined by Lin et al. [15]. The composition of this medium was as follows (g/L): 10
124
colloidal chitin; 4.2 ammonium sulfate; 6.9 monobasic sodium phosphate; 2
125
monobasic potassium phosphate; 0.3 magnesium sulfate with 62.5 mL of salt
126
solution. The medium was adjusted to pH 5.0 and sterilized by autoclave.
127
Two forms of the fungi were tested, mycelium and spores. For the first case a disc
128
of 1 cm diameter, saturated with T. harzianum mycelia from a PDA plate and
129
incubated for 72 h, was added to a flask with the production medium. In the case of
130
spores, the initial concentration in each flask was adjusted to 1x106 spores/mL.
131
Both were cultured at 28°C/120 rpm for 7 days. After this time, the supernatant was
132
recovered after centrifugation (3000 xg/10 min) and sterilized by filtration (0.22µm)
133
to obtain the crude enzyme. The experiments were carried out in triplicate.
134
2.3.
Obtaining chitosan variants
135
Three types of chitosan were used for production of COS: 1) Colloidal chitosan
136
(CC), solid chitosan (CS) and chitosan solution (DC). Colloidal chitosan (CC) was
137
prepared according to our previous protocol, using 85% phosphoric acid and
138
resuspension with ethanol [13]. Stock chitosan solution (DC, 50 g/L) was prepared
139
with solid low molecular weight chitosan (Sigma Aldrich St. Louis, MO, USA) in
140
0.1% acetic acid [18]. In the case of CS, solid low molecular weight chitosan
141
without any processing was used (Sigma Aldrich St. Louis, MO, USA).
142
2.4.
COS production
143
The solid substrates were suspended in 0.1 M acetate buffer pH 4.0 at a
144
concentration of 11 g/L. DC substrate was used at the same concentration.
145
Substrates were mixed with the sterile crude enzymes obtained from spores or
146
from mycelia (section 2.3) separately, at a substrate/enzyme ratio of 10:1 (w/v or
147
v/v depending on the case). The mixtures were incubated for 7 days at 42°C and
148
the concentration of reducing sugar was monitored by the DNS method to select
149
the best system for the production of COS [19].
150
2.5.
Chromatographic analysis
151
For the chromatographic analysis, samples were concentrated 20 times by
152
lyophilization. TLC and HPLC techniques for qualitative and quantitative analysis
153
were carried out. In the first case, 20 µL of the samples were placed on 10x5 cm
154
silica gel plates and a water-propanol-ammonium hydroxide solution (7:3:1) was
155
used as the mobile phase [13]. COS standard (C3-C7 Carbosynth, Berkshire,
156
United Kingdom) and 0.1 M glucosamine were used as standards. The plates were
157
treated with an alcohol-sulfuric acid solution (10:1) and heated at 150°C for COS
158
observation. For HPLC, a Tec Agilent 1200 series (Agilent Technologies, Santa
159
Clara, CA) coupled to an amino column (Hypersil APS-2 brand Thermo Fisher
160
Scientific; 4.6 × 150 mm) was employed. An acetonitrile–water (70:30) mixture was
161
used as the mobile phase with a flow rate of 1.5 mL/min. 10 µL of the concentrated
162
sample was injected for the quantification of COS, each peak was interpolated in a
163
standard curve using the same standards as in TLC [13].
164
2.6.
Antimicrobial effect
165
The antimicrobial effect was measured using a growth inhibition test by plate
166
diffusion. The methicillin-resistant Staphylococcus aureus (MRSA) USA 300 (Gram
167
+); Escherichia coli ATTCCK12 (Gram -), Ustilago maydis FB2 ATCC201384
168
(Basidiomycete) and Candida albicans ATCC10231 (Ascomycete) were used to
169
test the effectiveness of the COS mixture in inhibiting the growth of bacteria and
170
yeasts. For bacteria, nutritive standard agar (Becton Dickinson Franklin Lakes, NJ,
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USA) was used; in the case of yeasts, YPD plates were prepared (1% yeast
172
extract, 0.15% ammonium nitrate, 0.25% Bacto peptone, 1% glucose and 2% agar,
173
pH 6.8). A cell suspension of each microorganism was prepared from a liquid pre-
174
inoculum and adjusted to number 1 on the McFarland scale (approximately, 0.25
175
UDO) and a massive plating was carried out by immersion of the microorganisms
176
in the medium. COS mixture (10, 5, 2.5, 1.25, 0.70 mg/mL) was placed on the
177
plates directly and incubated at 28°C (yeasts) and 37°C (bacteria) for 12-18 h. The
178
experiments were done in triplicate and the inhibitory effect was evaluated
179
qualitatively. To determine the EC50 and the minimum inhibitory concentration
180
(MIC) of the COS mixture, concentrations from 1 to 10 mg/mL were evaluated,
181
according to the method reported by Olicón-Hernández et al. [18]. The growth of
182
the strains was expressed as % of the optical density (O.D.) at 600 nm of the
183
control (cells incubated in the absence of the COS mixture) after 24 h of incubation
184
(37°C/120 rpm).
185
2.7.
Anticancer effect
186
To determine the cytotoxic effect of COS on cervical cancer cells, we performed a
187
cell proliferation assay according to the protocol described by Arora et al. [20]. 3 x
188
104 HeLa cells were cultured in 6-well plates under standard conditions [Eagle's
189
Minimum Essential Medium, 10% (v / v) FBS obtained from Sigma-Aldrich at 37°C
190
in at 5% CO2 incubator]. After 24 hours of growth, the cells were transferred to
191
fresh medium and COS was added at different concentrations (control, 4, 6 and 8
192
mg/mL) and incubated for 48 hrs. After the treatment period, the medium was
193
replaced, and the cells were washed with PBS. Next, the cells were fixed with
194
0.75% crystal violet (m/v) in ethanol for 60 min. Subsequently, the images of the
195
cultures were taken at different scales (5X, 10X, 20X) with the help of an inverted
196
microscope. Finally, the cells were washed with a 1% SDS solution and the
197
absorbance was measured at 570 nm.
198
2.8.
COS purification
199
For purification of the components of the COS mixture, a biogel p-4 (Biorad
200
Hercules, California, USA) was placed into a glass column (2x30 cm) with distilled
201
water as the eluent, with a flow rate of 0.14 mL/min. Five mL of concentrated COS
202
mixture was placed on the top of the column and fractions of 400 µL were
203
collected. The concentration of reducing sugars in each fraction was determined by
204
the DNS method [19], and the fractions with a higher concentration were assessed
205
by TLC under the conditions described above.
206
3. Results
207
3.1.
Comparison of COS production systems
208
The kinetics of COS production using the six proposed systems is shown in Figure
209
1A. In all cases, the maximum production of reducing sugar was achieved at 7
210
days of incubation, with the colloidal chitosan with spores crude enzyme (CCS)
211
being the system with the higher production. The lowest production was observed
212
with COS produced with chitosan solution and the mycelium of T. harzianum.
213
Regarding the other COS production systems, no significant differences were
214
observed. For ease of handling, it was decided to analyze the composition of the
215
mixtures of the samples that used spores for the production of COS. The TLC
216
analysis of these samples is shown in Figure 1B. Interestingly, the CCS system
217
was composed exclusively by glucosamine and was not useful for the production of
218
COS, even though it was the system that presented the best performance in the
219
initial exploration. In contrast, solid chitosan with spores (CSS) provided the best
220
COS production profile. According to the results, the CSS system was selected for
221
the production of COS and used for the subsequent experiments.
222
3.2.
Quantification of COS by HPLC
223
The quantitative analysis of the COS mixture from the CSS system is shown in
224
Figure 2. The chromatogram shows the signal of six COS, including the monomer
225
and dimer of glucosamine. C3 and C4 signals were partially overlapping and no
226
signal was observed for C7. The main component of the mixture was glucosamine,
227
represented by more than 80%, followed by dimers, trimers and pentamers
228
respectively.
229 230
3.3.
COS mixture inhibited the growth of bacteria and changed the
morphology of yeast
231
The COS mixture affected the growth of bacteria at concentrations of 2.5 and 10
232
mg/mL (Figure 3). E. coli was the most sensitive bacteria, being inhibited by 2.5
233
mg/mL (Figure 3B). On the other hand, S. aureus was inhibited only at the highest
234
concentrations of the COS mixture (Figure 3A). However, it is important to mention
235
that this is an MRSA strain and, outstandingly, the COS mixture showed an
236
important inhibitory effect at the concentrations mentioned above. The MIC and
237
EC50 are shown in Figure 4. The results corroborate that E. coli was more sensitive
238
than S. aureus (approximately EC50 = 5.1 mg/mL and MIC = 9.6 mg/mL; EC50 = 9.4
239
mg/mL and MIC = 16.9 mg/mL respectively)
240
In the case of yeasts, inhibition of the growth was null at all tested concentrations;
241
however, at 10 mg/mL both yeasts modified their cellular morphology, resulting in a
242
change in their colony morphology on the plate (Figure 3C and 3D), with the
243
strongest change in U. maydis. Since yeasts were not sensitive to COS mixture,
244
MIC and EC50 were not determined.
245
3.4.
Anticancer effect
246
Cell proliferation in HeLa cells was affected by COS at 48 hrs. There was a
247
modification in the morphology of the HeLa cells at all the concentrations tested,
248
with deformation of the cell body, a reduction in the size and number of the cells,
249
and the presence of extracellular bodies being observed (Figure 5). The greatest
250
effect on cell proliferation was obtained at a concentration of 8 mg/mL, with a
251
decrease close to 40% (p<0.05). At 4 and 6 mg / mL, cell proliferation decreased
252
23% and 32%, respectively (Figure 6).
253
3.5.
Partial purification system of COS
254
An analysis of the fractions obtained by the purification of the COS mixture is
255
shown in Figure 7A. Six, single-well, defined peaks (p1-p4 and p7-p8) were
256
detected by the DNS method and 1 wide peak (p4-p5) was divided into two for the
257
TLC experiment. The highest absorbance values corresponded to the last signals
258
(p7-p8), in contrast to the first four that had the weakest signals. According to TLC
259
plates (Figure 7B), the fractions p1-p2 (Figure 7B-1) did not have well-defined COS
260
and the concentration of the compounds was the lowest; the p3 and p6 fractions
261
had the dimer (Figure 7B-2 and -3); p4 and p5 contained a COS mixture that
262
included the dimer but not the monomer (Figure 7B-2); and p7-p8 was formed
263
exclusively by glucosamine (Figure 7B-2). With this method it was possible to
264
purify the monomer and the dimer from the mixture of COS, but it was not possible
265
to separate the rest of the COS individually.
266
4. Discussion
267
COS have interesting clinical applications that have promoted the development of
268
diverse production systems for these molecules. In this work, we showed that the
269
accessibility and presentation of the substrate affects COS profile, even to the
270
point of producing only the monomer. In this context, Santo-Mariano [21] proposed
271
the continuous production of COS by α-amylase from Bacillus amylolyquefaciens in
272
a dual-reactor system testing 3 different chitosan variants. In this case, QS1
273
chitosan (MW 95.5 KDa, 81% degree of deacetylation, DD); CHIT100 chitosan
274
(MW 100–300 KDa, DD≥90%) and CHIT600 chitosan (MW 600–800 KDa.
275
DD≥90%) were the substrates. The profile of COS changed according to the
276
substrate; for example, QS1 produced an acetylated COS mixture, as a
277
consequence of the low degree of deacetylation (DD) of the substrate (81%). It has
278
been seen that this type of COS has a smaller effect on human health. In addition,
279
COS profile was similar with the other substrates, although, a higher concentration
280
of glucosamine was obtained with CHIT600 [21]. This result is similar to our
281
findings with colloidal chitosan.
282
According to our results, the best substrate for the production of COS was solid
283
crystalline chitosan, which is even more efficient because it can be used directly.
284
This result contrasts with that reported by Nidheesh et al., who found a higher
285
production of COS with colloidal chitosan (4.43 mM of COS) as compared to
286
crystalline chitosan (1.7 mM) after 24 h of hydrolysis using the Purpureocillium
287
lilacinum CFRNT12 chitosanase [22]. In our research group, the use of colloidal
288
chitosan for the production of COS using Bacillus thuriengiensis endochitosanase
289
was reported, obtaining a mixture of COS from C1 to C6 with high production
290
yields [13]. Taken together, these results indicate that the enzymatic system of
291
each microorganism is different and independently coupled to each substrate. In
292
addition, the absence of a chemical pretreatment to obtain the colloidal chitosan is
293
a favorable point for the use of Trichoderma harzianum enzymes.
294
The characteristics of the original polymer could be crucial for high yields of
295
production and/or to increase the biological effect. For example, chitosan oligomers
296
produced by the chitinase of Serratia proteamaculans (wild-type and mutant),
297
employing chitosan with 35 and 61% of acetylation (DA) as substrates, induced an
298
oxidative burst in rice cell cultures [23]. In this case, the COS mixture was more
299
effective when the substrate had more DA; however, the degree of polymerization
300
of COS (DP) is also an important parameter, since the shorter COS were the worst
301
inducers of the defense response in plants [23].
302
Lin et al. previously reported the use of T. harzianum for the production of COS,
303
observing a maximum global concentration of reducing sugars of approximately 70
304
mmol/L using β-chitosan. However, with this method it is not possible to distinguish
305
the percentage that corresponds to COS, since the medium contains other
306
elements that react with the DNS reagent. In addition, a complete characterization
307
of the COS polymerization profile and its quantification was not performed [15]. In
308
the literature, different yields have been reported in the production of COS, and
309
these depend on the type of enzyme, microorganisms, substrate, and/or production
310
system [14].
311
Different chitosan enzymatic systems have been tested. For example, the
312
production of low molecular weight chitosan derivatives was optimized by surface
313
response surface methodology using commercial papain (protease). In this case, a
314
kinetic characterization of the products of the hydrolysis showed that the initial
315
concentration of chitosan is an important parameter for COS production, since
316
papain was inhibited when the chitosan concentration was above 8 g/L [24]. A
317
recent approach to COS production uses low molecular weight derivatives of chitin
318
which deacetylate with recombinant enzymes from Rhizobium sp. and Vibrio
319
cholerae to obtain the COS mixture; this system has a better control of the degree
320
of deacetylation and polymerization of the resulting mixture [25].
321
Here we reported the sensitivity of Gram (+) and (-) bacteria against the COS
322
mixture, with E. coli being the most affected by the presence of the compounds.
323
This result is consistent the higher minimum inhibitory concentration (MIC) of COS
324
in gram positive bacteria than in gram negative bacteria observed by Li et al. A
325
point to note is that they reported that the growth of S. aureus was not inhibited by
326
COS, whereas we found the opposite [15]. However, in other works it was found
327
sensitivity of this bacterium to the mixture of COS [26-28]. Another important point
328
is that our COS mixture is effective against the growth of a strain of S. aureus type
329
MRSA (Methicillin-resistant strain), which opens the possibility of using COS as an
330
alternative treatment for strains resistant to antibiotics. The mode of action of COS
331
mixture against bacteria is unclear, however, the activity depends on several
332
factors such as degree of polymerization and deacetylation, type of microorganism
333
and physico-chemical properties of the cell wall. The most accepted mode of action
334
of the antibacterial activity is related to the free amino group and the positive
335
charge of COS that can alter cell membrane permeability causing the leakage of
336
cell constituents that finally leads to the death of bacteria. The charge distribution
337
of bacterial cell wall seems to play a main role for the antibacterial activities of the
338
positively charged COS. Bacterial cell wall has a negative charge distribution. In
339
Gram-negative bacteria is higher than in Gram-positive bacteria. Therefore, the
340
adsorption of the positive charged COS on the surface is higher in Gram-negative
341
bacteria than in Gram-positive bacteria. This explains the reason why most Gram-
342
negative bacteria are more sensitive to COS mixtures. [14, 18].
343
In the case of the antifungal effect, it was demonstrated that COS with more than
344
20 repetitions of the monomer inhibited the growth fungi (Botrytris cinerea and
345
Mucor piriformis) better than those that have a lower degree of polymerization,
346
such as those obtained in this work (3-6 units), which would explain the null effect
347
of our mixture on the yeasts [29]. In agreement with this conclusion, chitosan
348
induced more significant damage in U. maydis structure compared to the low
349
molecular weight derivative oligochitosan [18]. It was reported that the COS
350
interferes with the synthesis of adhesive compounds and biofilm precursor, which,
351
added to its polycationic nature, would explain the cell aggregation and
352
morphological changes as consequence of the interaction with the COS mixture
353
[30].
354
As anticancer compounds, it has been reported that COS can interfere with cell
355
proliferation and morphology and the metastasis of various tumor lines [31]. Here,
356
we demonstrated that the COS mixture produced by T. harzianum chitinase,
357
changed the morphology of HeLa cells and significantly reduced the survival of
358
cells. This result is consistent with that reported by de Asis et al. where a COS
359
mixture obtained from the fungus Metarhizium Anisopliae reduced the proliferation
360
of HeLa cells up to 60%, but did not affect HepG2 hepatocarcinoma cells (ATCC
361
HB-8065) [32]. In contrast, Ronghua Huang et al. reported that a standard mixture
362
of COS did not affect the viability of HeLa cells. However, modification of the COS
363
charge by chemical inclusions improved the anticancer capacity of the compounds
364
[33]. de Assis et al. observed that different effects on HeLa cells proliferation
365
depended on the COS composition, suggesting that a combination of COS
366
products may be essential for developing antineoplastic drugs [32]. The exact
367
mechanism against the proliferation of cancer cells is unknown, but may be
368
associated with the electrostatic charges of COS, changes in the permeability of
369
tumor cells and regulation of the expression of tumor factors such as
370
metalloproteinase-9 or/and vascular endothelial growth factor [34].
371
Although our results are promising, important improvements in the method of
372
production and further experiments on the anticancer and antimicrobial effects
373
should be implemented for the potential clinical applications.
374
5. Conclusions
375
The chitin-hydrolytic system of T. harzianum produces a chitosan-oligosaccharide
376
mixture composed from monomers to hexamers/heptamers, with solid crystalline
377
chitosan and fungi spores providing the best conditions for COS production. The
378
COS mixture showed a strong growth inhibition effect against Gram (-) and
379
antibiotic resistant Gram (+) bacteria, but was ineffective in inhibiting yeast growth.
380
The COS mixture has potential effective anticancer effects against cervical cancer
381
HeLa cells, but it will be essential to test its effects against other cell lines.
382 383
Acknowledgments and funding sources
384
This work was supported by CONACyT grants 254904 (JPP) and 256502 (GGS),
385
SIP project 20190200 (GGS) and PAPIIT-DGAPA project IN222117 (JPP). The
386
first author (DROH) want to thank Dirección General de Asuntos del Personal
387
Académico (DGAPA) program of Universidad Nacional Autónoma de México for
388
the support of the postdoctoral fellowship.
389 390
Figure captions:
391
Figure 1. Analysis of COS production by T. harzianum. A) Reducing sugar
392
quantification in the production systems of COS. Reducing sugars were observed
393
over 198 h. CC=colloidal chitosan; CS=solid chitosan; DC=chitosan solution;
394
S=fungus spores; M=fungus mycelium. B) TLC of COS mixture from the highest
395
reducing sugar systems. GlcN=monomer (glucosamine); C3-C7=trimer to
396
heptamer. Only the final samples of fungus spore systems were analyzed.
397
Figure 2. Quantification of COS mixture. The HPLC analysis showed a composition
398
with mixture of monomers to hexamers. The most abundant component was
399
glucosamine, followed by dimers, trimers and pentamers. C1-C6=Monomer-
400
Hexamer
401
Figure 3. Antimicrobial inhibitory growth effect of COS mixture. A) Staphylococcus
402
aureus; B) Escherichia coli; C) Candida albicans; D) Ustilago maydis.
403
Concentration tested (mg/mL) 1=10; 2=5; 3=2.5; 4=1.25 and 5=0.70.
404
405
Figure 4. Bacterial growth in different COS mixture concentrations. The growth was
406
expressed as % of the control O.D.600nm after 24 h of incubation at 37 °C. MIC (red
407
line) and EC50 (blue line).
408
Figure 5. Proliferation of HeLa cells in the presence of COS at different
409
concentrations. Changes in the structure and number of HeLa cells were observed
410
under treatment at all concentrations. Cells grown without COS were used as
411
control.
412
Figure 6. Cytotoxic effect of COS on cervical cancer cells. Cells grown without
413
COS were used as control. (*) represents statistically significant differences. The
414
statistical test used for comparison was the student t-test for unpaired samples with
415
Welch correction, with p = 0.048.
416
Figure 7. Purification profile of COS using biogel p-4. A) Elution peaks of COS
417
detected by DNS method. B) TLC of the selected peaks. 1) p1-p2; 2) p3-p5; 3) p6-
418
p8. GlcN=glucosamine; C3-C7= trimer-heptamer.
419 420 421
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Highlights •
Trichoderma harzianum chitinase is a potential system for COS production
•
T. harzianum enzymes produces a COS mixture from monomers to hexamers.
•
The maximum yield of COS was obtained using solid crystal chitosan and fungus spores
•
COS mixture has an effective antimicrobial and anticancer effect
Conflict of Interest Statement
The author declares that the research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.
S0008-6215(19)30494-X
DOI:
https://doi.org/10.1016/j.carres.2019.107836
Reference:
CAR 107836
To appear in:
Carbohydrate Research
Received Date: 22 August 2019 Revised Date:
23 September 2019
Accepted Date: 15 October 2019
Please cite this article as: Olicó.-Herná. Dario Rafael, Z.-G. Luis Fernándo, P.-T. Abraham, Vá.Landaverde. Pedro Alberto, Guerra.-Sá. Guadalupe, P.J. Pablo, Production of chitosan-oligosaccharides by the chitin-hydrolytic system of Trichoderma harzianum and their antimicrobial and anticancer effects, Carbohydrate Research (2019), doi: https://doi.org/10.1016/j.carres.2019.107836. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
1
Production of chitosan-oligosaccharides by the chitin-hydrolytic system of
2
Trichoderma harzianum and their antimicrobial and anticancer effects
3 4
Olicón-Hernández Dario Rafaela; Zepeda-Giraud Luis Fernándob; Pedroza-Torres
5
Abrahamc; Vázquez-Landaverde Pedro Albertod; Guerra-Sánchez Guadalupeb;
6
Pardo Juan Pabloa*.
7 8
a
9
de Bioquímica. Laboratorio 7. Circuito Interior s/n, Ciudad Universitaria CP 04510,
Universidad Nacional Autónoma de México. Facultad de Medicina. Departamento
10
Ciudad de México, México.
11
b
12
Departamento de Microbiología. Laboratorio de bioquímica y biotecnología de
13
hongos. Carpio y Plan de Ayala s/n. Santo Tomas, Miguel Hidalgo. CP 11350,
14
Ciudad de México, México.
15
c
16
Hereditario. Avenida San Fernando 22, Belisario Domínguez Secc XVI, CP 14080
17
Ciudad de México, México.
18
d
19
Tecnología Avanzada, Unidad Querétaro. Cerro Blanco 141. Colinas del
20
Cimatario. CP 76090 Querétaro, México.
Instituto Politécnico Nacional. Escuela Nacional de Ciencias Biológicas.
Cátedra CONACYT-Instituto Nacional de Cancerología. Clínica de Cáncer
Instituto Politécnico Nacional. Centro de Investigación en Ciencia Aplicada y
21 22
*Corresponding author:
23
Pardo Juan Pablo
24
Universidad Nacional Autónoma de México. Facultad de Medicina. Departamento
25
de Bioquímica. Laboratorio 7. Circuito Interior s/n, Ciudad Universitaria CP 04510,
26
Ciudad de México, México. Phone number: (+52) 555623 2175. Email:
27
[email protected]
28
Abstract
29
Chitosan-oligosaccharides (COS) are low-molecular weight chitosan derivatives
30
with interesting clinical applications. The optimization of both COS production and
31
purification is an important step in the design of an efficient production system and
32
for the exploration of new COS applications. Trichoderma harzianum is an
33
innocuous biocontrol agent that represents a novel biotechnological tool due to the
34
production of extracellular enzymes, including those that produce a COS mixture.
35
In this work, we propose different systems for the production of COS using the T.
36
harzianum chitinolitic system. A complete qualitative and quantitative analysis of a
37
partially purified COS mixture were performed. Also, an evaluation of the
38
anticancer and antimicrobial effects of the COS mixture was carried out. Three
39
chitosan variants (colloidal, solid and solution) and two fungus stages (spores and
40
mycelia) were tested for COS production. The best system consisted of the
41
interaction of the solid chitosan and the fungal spores, producing a COS mixture
42
containing species from the monomer to the hexamer, in a concentration range of
43
7 to 238 mg/mL, according to chromatographic analysis. The proposed purification
44
method isolated the monomer and the dimer from the COS mixture. Moreover, the
45
COS mixture has an inhibitory effect on the growth of bacteria and changes the
46
morphology of yeasts. As anticancer compounds, COS inhibited the growth of
47
cervical cancer cells at concentration of 4 mg/mL and significantly reduced the
48
survival rate of the cells. In conclusion, T. harzianum proved to be an efficient
49
system for COS mixture production.
50
Key words: Chitosan-oligosaccharides; Trichoderma harzianum; antimicrobial;
51
anticancer
52 53 54 55 56
57
1. Introduction
58
Trichoderma harzianum is a filamentous ascomycete used as a biocontrol element
59
and biotechnological tool that has gained interest in recent years [1, 2]. This fungus
60
is a cosmopolitan microorganism, with applications in the field of agriculture, being
61
part of commercial mixtures used for composting and involved in the control of
62
pests in crops of interest [3]. The applications of this fungus are not limited to
63
agricultural topics, since it also has the ability to degrade different substrates to
64
produce extracellular enzymes of industrial interest, and has been used for the
65
manufacture of nanoparticles with antibiotic action and the production of biofuels
66
[2, 4-6].
67
It has been reported that T. harzianum has a powerful inhibitory effect against
68
phytopathogenic fungi, with mycoparasitism as one of the most important
69
mechanisms associated with this effect; however, the production of antibiotic
70
molecules as well as the secretion of hydrolytic enzymes that attack the cell wall of
71
different fungi also have an important role in biocontrol behavior, with the group
72
extracellular chitinases being one of the most interesting [7]. In this context, 331
73
enzymes with chitinase activity were reported in the uniprot database for T.
74
harzianum and four of them were identified and characterized as endochitinases
75
(https://www.uniprot.org/) [8]. Possibly, T. harzianum has one of the most versatile
76
"chitinomes" in nature since it is known that some strains have changes in their
77
gene architecture, signal peptide, domain organization and molecular weight in
78
chitinase production [9].
79
Chitinases (EC 3.2.1.14) are endo- and exo- enzymes that degrade chitin, a dense
80
and crystalline N-acetyl-glucosamine-polymer present in insects and crustaceans.
81
In fungi, chitinases contribute to morphogenetic, nutritional and pathogenic
82
processes, including spore germination, hyphal branching, autolysis and
83
mycoparasitic interactions [10]. From a biotechnological point of view, chitinases
84
are used either as bioinsecticides or to obtain bioactive derivatives from chitin
85
and/or chitosan (deacetylated derivative of chitin).
86
Chitosan-oligosaccharides (COS) are low-molecular-weight chains of 6-10
87
glucosamine repeats derived from the action of chitinases on chitosan [11]. COS
88
are potential therapeutic compounds owing to their importance for human health,
89
for example via their proposed use as antibiotics, anticancer and anti-cholesterol
90
molecules [11, 12]. COS are produced by a variety of extracellular enzymes,
91
including chitosanases, chitinases, papain and cellulases [13, 14]; however, many
92
of these only explore production with a single substrate.
93
To our knowledge, there is only one report on the use of chitinase from T.
94
harzianum for the production of COS [15]. In this study, COS mixture was obtained
95
from α and/or β-chitosan and the mixture was effective against bacteria. However,
96
in this work the composition of the COS mixture was not completely identified and
97
the influence of variants of the substrate or the stage within the cell cycle of the
98
fungus were not tested.
99
The goals of this work were as follows: 1) to evaluate six different systems for the
100
production of COS using the chitinase from T. harzianum and the combination of 3
101
variants of chitosan (colloidal, solid and as dilution) and 2 cell cycle stages of the
102
fungus (spores and mycelia); 2) to identify and quantify this COS mixture by
103
chromatographic techniques; 3) to develop a partial purification system and, 4) to
104
search for clinical applications of COS as antimicrobial and anticancer agents.
105
2. Materials and methods
106
2.1.
Strain and culture conditions
107
Trichoderma harzianum strain T1 was isolated from contaminated soil and belongs
108
to a collection of strains in the laboratorio de bioquímica y biotecnología de hongos
109
of ENCB-IPN, México. This strain exhibited tolerance in the biodegradation of
110
petroleum hydrocarbons according to the results reported by Argumedo-Delira et
111
al. [16].
112
Commercial Potato Dextrose Agar and Broth (PDA and PDB, Becton Dickinson
113
Franklin Lakes, NJ, USA) were used for the for the maintenance and storage of the
114
T1 strain.
115
To obtain mycelium, discs of 1 cm diameter were cut from PDA plates with
116
previous growth and placed on fresh plates. Discs were cultured at 28°C under
117
static conditions until the mycelium occupied the entire plate. The collection of
118
spores was obtained by mechanical processes using plates saturated with
119
mycelium, according to our previous protocol and adjusted by the Neubauer
120
chamber method [17].
121
2.2.
Chitinase induction
122
For chitin induction, a production medium was designed based on the method
123
outlined by Lin et al. [15]. The composition of this medium was as follows (g/L): 10
124
colloidal chitin; 4.2 ammonium sulfate; 6.9 monobasic sodium phosphate; 2
125
monobasic potassium phosphate; 0.3 magnesium sulfate with 62.5 mL of salt
126
solution. The medium was adjusted to pH 5.0 and sterilized by autoclave.
127
Two forms of the fungi were tested, mycelium and spores. For the first case a disc
128
of 1 cm diameter, saturated with T. harzianum mycelia from a PDA plate and
129
incubated for 72 h, was added to a flask with the production medium. In the case of
130
spores, the initial concentration in each flask was adjusted to 1x106 spores/mL.
131
Both were cultured at 28°C/120 rpm for 7 days. After this time, the supernatant was
132
recovered after centrifugation (3000 xg/10 min) and sterilized by filtration (0.22µm)
133
to obtain the crude enzyme. The experiments were carried out in triplicate.
134
2.3.
Obtaining chitosan variants
135
Three types of chitosan were used for production of COS: 1) Colloidal chitosan
136
(CC), solid chitosan (CS) and chitosan solution (DC). Colloidal chitosan (CC) was
137
prepared according to our previous protocol, using 85% phosphoric acid and
138
resuspension with ethanol [13]. Stock chitosan solution (DC, 50 g/L) was prepared
139
with solid low molecular weight chitosan (Sigma Aldrich St. Louis, MO, USA) in
140
0.1% acetic acid [18]. In the case of CS, solid low molecular weight chitosan
141
without any processing was used (Sigma Aldrich St. Louis, MO, USA).
142
2.4.
COS production
143
The solid substrates were suspended in 0.1 M acetate buffer pH 4.0 at a
144
concentration of 11 g/L. DC substrate was used at the same concentration.
145
Substrates were mixed with the sterile crude enzymes obtained from spores or
146
from mycelia (section 2.3) separately, at a substrate/enzyme ratio of 10:1 (w/v or
147
v/v depending on the case). The mixtures were incubated for 7 days at 42°C and
148
the concentration of reducing sugar was monitored by the DNS method to select
149
the best system for the production of COS [19].
150
2.5.
Chromatographic analysis
151
For the chromatographic analysis, samples were concentrated 20 times by
152
lyophilization. TLC and HPLC techniques for qualitative and quantitative analysis
153
were carried out. In the first case, 20 µL of the samples were placed on 10x5 cm
154
silica gel plates and a water-propanol-ammonium hydroxide solution (7:3:1) was
155
used as the mobile phase [13]. COS standard (C3-C7 Carbosynth, Berkshire,
156
United Kingdom) and 0.1 M glucosamine were used as standards. The plates were
157
treated with an alcohol-sulfuric acid solution (10:1) and heated at 150°C for COS
158
observation. For HPLC, a Tec Agilent 1200 series (Agilent Technologies, Santa
159
Clara, CA) coupled to an amino column (Hypersil APS-2 brand Thermo Fisher
160
Scientific; 4.6 × 150 mm) was employed. An acetonitrile–water (70:30) mixture was
161
used as the mobile phase with a flow rate of 1.5 mL/min. 10 µL of the concentrated
162
sample was injected for the quantification of COS, each peak was interpolated in a
163
standard curve using the same standards as in TLC [13].
164
2.6.
Antimicrobial effect
165
The antimicrobial effect was measured using a growth inhibition test by plate
166
diffusion. The methicillin-resistant Staphylococcus aureus (MRSA) USA 300 (Gram
167
+); Escherichia coli ATTCCK12 (Gram -), Ustilago maydis FB2 ATCC201384
168
(Basidiomycete) and Candida albicans ATCC10231 (Ascomycete) were used to
169
test the effectiveness of the COS mixture in inhibiting the growth of bacteria and
170
yeasts. For bacteria, nutritive standard agar (Becton Dickinson Franklin Lakes, NJ,
171
USA) was used; in the case of yeasts, YPD plates were prepared (1% yeast
172
extract, 0.15% ammonium nitrate, 0.25% Bacto peptone, 1% glucose and 2% agar,
173
pH 6.8). A cell suspension of each microorganism was prepared from a liquid pre-
174
inoculum and adjusted to number 1 on the McFarland scale (approximately, 0.25
175
UDO) and a massive plating was carried out by immersion of the microorganisms
176
in the medium. COS mixture (10, 5, 2.5, 1.25, 0.70 mg/mL) was placed on the
177
plates directly and incubated at 28°C (yeasts) and 37°C (bacteria) for 12-18 h. The
178
experiments were done in triplicate and the inhibitory effect was evaluated
179
qualitatively. To determine the EC50 and the minimum inhibitory concentration
180
(MIC) of the COS mixture, concentrations from 1 to 10 mg/mL were evaluated,
181
according to the method reported by Olicón-Hernández et al. [18]. The growth of
182
the strains was expressed as % of the optical density (O.D.) at 600 nm of the
183
control (cells incubated in the absence of the COS mixture) after 24 h of incubation
184
(37°C/120 rpm).
185
2.7.
Anticancer effect
186
To determine the cytotoxic effect of COS on cervical cancer cells, we performed a
187
cell proliferation assay according to the protocol described by Arora et al. [20]. 3 x
188
104 HeLa cells were cultured in 6-well plates under standard conditions [Eagle's
189
Minimum Essential Medium, 10% (v / v) FBS obtained from Sigma-Aldrich at 37°C
190
in at 5% CO2 incubator]. After 24 hours of growth, the cells were transferred to
191
fresh medium and COS was added at different concentrations (control, 4, 6 and 8
192
mg/mL) and incubated for 48 hrs. After the treatment period, the medium was
193
replaced, and the cells were washed with PBS. Next, the cells were fixed with
194
0.75% crystal violet (m/v) in ethanol for 60 min. Subsequently, the images of the
195
cultures were taken at different scales (5X, 10X, 20X) with the help of an inverted
196
microscope. Finally, the cells were washed with a 1% SDS solution and the
197
absorbance was measured at 570 nm.
198
2.8.
COS purification
199
For purification of the components of the COS mixture, a biogel p-4 (Biorad
200
Hercules, California, USA) was placed into a glass column (2x30 cm) with distilled
201
water as the eluent, with a flow rate of 0.14 mL/min. Five mL of concentrated COS
202
mixture was placed on the top of the column and fractions of 400 µL were
203
collected. The concentration of reducing sugars in each fraction was determined by
204
the DNS method [19], and the fractions with a higher concentration were assessed
205
by TLC under the conditions described above.
206
3. Results
207
3.1.
Comparison of COS production systems
208
The kinetics of COS production using the six proposed systems is shown in Figure
209
1A. In all cases, the maximum production of reducing sugar was achieved at 7
210
days of incubation, with the colloidal chitosan with spores crude enzyme (CCS)
211
being the system with the higher production. The lowest production was observed
212
with COS produced with chitosan solution and the mycelium of T. harzianum.
213
Regarding the other COS production systems, no significant differences were
214
observed. For ease of handling, it was decided to analyze the composition of the
215
mixtures of the samples that used spores for the production of COS. The TLC
216
analysis of these samples is shown in Figure 1B. Interestingly, the CCS system
217
was composed exclusively by glucosamine and was not useful for the production of
218
COS, even though it was the system that presented the best performance in the
219
initial exploration. In contrast, solid chitosan with spores (CSS) provided the best
220
COS production profile. According to the results, the CSS system was selected for
221
the production of COS and used for the subsequent experiments.
222
3.2.
Quantification of COS by HPLC
223
The quantitative analysis of the COS mixture from the CSS system is shown in
224
Figure 2. The chromatogram shows the signal of six COS, including the monomer
225
and dimer of glucosamine. C3 and C4 signals were partially overlapping and no
226
signal was observed for C7. The main component of the mixture was glucosamine,
227
represented by more than 80%, followed by dimers, trimers and pentamers
228
respectively.
229 230
3.3.
COS mixture inhibited the growth of bacteria and changed the
morphology of yeast
231
The COS mixture affected the growth of bacteria at concentrations of 2.5 and 10
232
mg/mL (Figure 3). E. coli was the most sensitive bacteria, being inhibited by 2.5
233
mg/mL (Figure 3B). On the other hand, S. aureus was inhibited only at the highest
234
concentrations of the COS mixture (Figure 3A). However, it is important to mention
235
that this is an MRSA strain and, outstandingly, the COS mixture showed an
236
important inhibitory effect at the concentrations mentioned above. The MIC and
237
EC50 are shown in Figure 4. The results corroborate that E. coli was more sensitive
238
than S. aureus (approximately EC50 = 5.1 mg/mL and MIC = 9.6 mg/mL; EC50 = 9.4
239
mg/mL and MIC = 16.9 mg/mL respectively)
240
In the case of yeasts, inhibition of the growth was null at all tested concentrations;
241
however, at 10 mg/mL both yeasts modified their cellular morphology, resulting in a
242
change in their colony morphology on the plate (Figure 3C and 3D), with the
243
strongest change in U. maydis. Since yeasts were not sensitive to COS mixture,
244
MIC and EC50 were not determined.
245
3.4.
Anticancer effect
246
Cell proliferation in HeLa cells was affected by COS at 48 hrs. There was a
247
modification in the morphology of the HeLa cells at all the concentrations tested,
248
with deformation of the cell body, a reduction in the size and number of the cells,
249
and the presence of extracellular bodies being observed (Figure 5). The greatest
250
effect on cell proliferation was obtained at a concentration of 8 mg/mL, with a
251
decrease close to 40% (p<0.05). At 4 and 6 mg / mL, cell proliferation decreased
252
23% and 32%, respectively (Figure 6).
253
3.5.
Partial purification system of COS
254
An analysis of the fractions obtained by the purification of the COS mixture is
255
shown in Figure 7A. Six, single-well, defined peaks (p1-p4 and p7-p8) were
256
detected by the DNS method and 1 wide peak (p4-p5) was divided into two for the
257
TLC experiment. The highest absorbance values corresponded to the last signals
258
(p7-p8), in contrast to the first four that had the weakest signals. According to TLC
259
plates (Figure 7B), the fractions p1-p2 (Figure 7B-1) did not have well-defined COS
260
and the concentration of the compounds was the lowest; the p3 and p6 fractions
261
had the dimer (Figure 7B-2 and -3); p4 and p5 contained a COS mixture that
262
included the dimer but not the monomer (Figure 7B-2); and p7-p8 was formed
263
exclusively by glucosamine (Figure 7B-2). With this method it was possible to
264
purify the monomer and the dimer from the mixture of COS, but it was not possible
265
to separate the rest of the COS individually.
266
4. Discussion
267
COS have interesting clinical applications that have promoted the development of
268
diverse production systems for these molecules. In this work, we showed that the
269
accessibility and presentation of the substrate affects COS profile, even to the
270
point of producing only the monomer. In this context, Santo-Mariano [21] proposed
271
the continuous production of COS by α-amylase from Bacillus amylolyquefaciens in
272
a dual-reactor system testing 3 different chitosan variants. In this case, QS1
273
chitosan (MW 95.5 KDa, 81% degree of deacetylation, DD); CHIT100 chitosan
274
(MW 100–300 KDa, DD≥90%) and CHIT600 chitosan (MW 600–800 KDa.
275
DD≥90%) were the substrates. The profile of COS changed according to the
276
substrate; for example, QS1 produced an acetylated COS mixture, as a
277
consequence of the low degree of deacetylation (DD) of the substrate (81%). It has
278
been seen that this type of COS has a smaller effect on human health. In addition,
279
COS profile was similar with the other substrates, although, a higher concentration
280
of glucosamine was obtained with CHIT600 [21]. This result is similar to our
281
findings with colloidal chitosan.
282
According to our results, the best substrate for the production of COS was solid
283
crystalline chitosan, which is even more efficient because it can be used directly.
284
This result contrasts with that reported by Nidheesh et al., who found a higher
285
production of COS with colloidal chitosan (4.43 mM of COS) as compared to
286
crystalline chitosan (1.7 mM) after 24 h of hydrolysis using the Purpureocillium
287
lilacinum CFRNT12 chitosanase [22]. In our research group, the use of colloidal
288
chitosan for the production of COS using Bacillus thuriengiensis endochitosanase
289
was reported, obtaining a mixture of COS from C1 to C6 with high production
290
yields [13]. Taken together, these results indicate that the enzymatic system of
291
each microorganism is different and independently coupled to each substrate. In
292
addition, the absence of a chemical pretreatment to obtain the colloidal chitosan is
293
a favorable point for the use of Trichoderma harzianum enzymes.
294
The characteristics of the original polymer could be crucial for high yields of
295
production and/or to increase the biological effect. For example, chitosan oligomers
296
produced by the chitinase of Serratia proteamaculans (wild-type and mutant),
297
employing chitosan with 35 and 61% of acetylation (DA) as substrates, induced an
298
oxidative burst in rice cell cultures [23]. In this case, the COS mixture was more
299
effective when the substrate had more DA; however, the degree of polymerization
300
of COS (DP) is also an important parameter, since the shorter COS were the worst
301
inducers of the defense response in plants [23].
302
Lin et al. previously reported the use of T. harzianum for the production of COS,
303
observing a maximum global concentration of reducing sugars of approximately 70
304
mmol/L using β-chitosan. However, with this method it is not possible to distinguish
305
the percentage that corresponds to COS, since the medium contains other
306
elements that react with the DNS reagent. In addition, a complete characterization
307
of the COS polymerization profile and its quantification was not performed [15]. In
308
the literature, different yields have been reported in the production of COS, and
309
these depend on the type of enzyme, microorganisms, substrate, and/or production
310
system [14].
311
Different chitosan enzymatic systems have been tested. For example, the
312
production of low molecular weight chitosan derivatives was optimized by surface
313
response surface methodology using commercial papain (protease). In this case, a
314
kinetic characterization of the products of the hydrolysis showed that the initial
315
concentration of chitosan is an important parameter for COS production, since
316
papain was inhibited when the chitosan concentration was above 8 g/L [24]. A
317
recent approach to COS production uses low molecular weight derivatives of chitin
318
which deacetylate with recombinant enzymes from Rhizobium sp. and Vibrio
319
cholerae to obtain the COS mixture; this system has a better control of the degree
320
of deacetylation and polymerization of the resulting mixture [25].
321
Here we reported the sensitivity of Gram (+) and (-) bacteria against the COS
322
mixture, with E. coli being the most affected by the presence of the compounds.
323
This result is consistent the higher minimum inhibitory concentration (MIC) of COS
324
in gram positive bacteria than in gram negative bacteria observed by Li et al. A
325
point to note is that they reported that the growth of S. aureus was not inhibited by
326
COS, whereas we found the opposite [15]. However, in other works it was found
327
sensitivity of this bacterium to the mixture of COS [26-28]. Another important point
328
is that our COS mixture is effective against the growth of a strain of S. aureus type
329
MRSA (Methicillin-resistant strain), which opens the possibility of using COS as an
330
alternative treatment for strains resistant to antibiotics. The mode of action of COS
331
mixture against bacteria is unclear, however, the activity depends on several
332
factors such as degree of polymerization and deacetylation, type of microorganism
333
and physico-chemical properties of the cell wall. The most accepted mode of action
334
of the antibacterial activity is related to the free amino group and the positive
335
charge of COS that can alter cell membrane permeability causing the leakage of
336
cell constituents that finally leads to the death of bacteria. The charge distribution
337
of bacterial cell wall seems to play a main role for the antibacterial activities of the
338
positively charged COS. Bacterial cell wall has a negative charge distribution. In
339
Gram-negative bacteria is higher than in Gram-positive bacteria. Therefore, the
340
adsorption of the positive charged COS on the surface is higher in Gram-negative
341
bacteria than in Gram-positive bacteria. This explains the reason why most Gram-
342
negative bacteria are more sensitive to COS mixtures. [14, 18].
343
In the case of the antifungal effect, it was demonstrated that COS with more than
344
20 repetitions of the monomer inhibited the growth fungi (Botrytris cinerea and
345
Mucor piriformis) better than those that have a lower degree of polymerization,
346
such as those obtained in this work (3-6 units), which would explain the null effect
347
of our mixture on the yeasts [29]. In agreement with this conclusion, chitosan
348
induced more significant damage in U. maydis structure compared to the low
349
molecular weight derivative oligochitosan [18]. It was reported that the COS
350
interferes with the synthesis of adhesive compounds and biofilm precursor, which,
351
added to its polycationic nature, would explain the cell aggregation and
352
morphological changes as consequence of the interaction with the COS mixture
353
[30].
354
As anticancer compounds, it has been reported that COS can interfere with cell
355
proliferation and morphology and the metastasis of various tumor lines [31]. Here,
356
we demonstrated that the COS mixture produced by T. harzianum chitinase,
357
changed the morphology of HeLa cells and significantly reduced the survival of
358
cells. This result is consistent with that reported by de Asis et al. where a COS
359
mixture obtained from the fungus Metarhizium Anisopliae reduced the proliferation
360
of HeLa cells up to 60%, but did not affect HepG2 hepatocarcinoma cells (ATCC
361
HB-8065) [32]. In contrast, Ronghua Huang et al. reported that a standard mixture
362
of COS did not affect the viability of HeLa cells. However, modification of the COS
363
charge by chemical inclusions improved the anticancer capacity of the compounds
364
[33]. de Assis et al. observed that different effects on HeLa cells proliferation
365
depended on the COS composition, suggesting that a combination of COS
366
products may be essential for developing antineoplastic drugs [32]. The exact
367
mechanism against the proliferation of cancer cells is unknown, but may be
368
associated with the electrostatic charges of COS, changes in the permeability of
369
tumor cells and regulation of the expression of tumor factors such as
370
metalloproteinase-9 or/and vascular endothelial growth factor [34].
371
Although our results are promising, important improvements in the method of
372
production and further experiments on the anticancer and antimicrobial effects
373
should be implemented for the potential clinical applications.
374
5. Conclusions
375
The chitin-hydrolytic system of T. harzianum produces a chitosan-oligosaccharide
376
mixture composed from monomers to hexamers/heptamers, with solid crystalline
377
chitosan and fungi spores providing the best conditions for COS production. The
378
COS mixture showed a strong growth inhibition effect against Gram (-) and
379
antibiotic resistant Gram (+) bacteria, but was ineffective in inhibiting yeast growth.
380
The COS mixture has potential effective anticancer effects against cervical cancer
381
HeLa cells, but it will be essential to test its effects against other cell lines.
382 383
Acknowledgments and funding sources
384
This work was supported by CONACyT grants 254904 (JPP) and 256502 (GGS),
385
SIP project 20190200 (GGS) and PAPIIT-DGAPA project IN222117 (JPP). The
386
first author (DROH) want to thank Dirección General de Asuntos del Personal
387
Académico (DGAPA) program of Universidad Nacional Autónoma de México for
388
the support of the postdoctoral fellowship.
389 390
Figure captions:
391
Figure 1. Analysis of COS production by T. harzianum. A) Reducing sugar
392
quantification in the production systems of COS. Reducing sugars were observed
393
over 198 h. CC=colloidal chitosan; CS=solid chitosan; DC=chitosan solution;
394
S=fungus spores; M=fungus mycelium. B) TLC of COS mixture from the highest
395
reducing sugar systems. GlcN=monomer (glucosamine); C3-C7=trimer to
396
heptamer. Only the final samples of fungus spore systems were analyzed.
397
Figure 2. Quantification of COS mixture. The HPLC analysis showed a composition
398
with mixture of monomers to hexamers. The most abundant component was
399
glucosamine, followed by dimers, trimers and pentamers. C1-C6=Monomer-
400
Hexamer
401
Figure 3. Antimicrobial inhibitory growth effect of COS mixture. A) Staphylococcus
402
aureus; B) Escherichia coli; C) Candida albicans; D) Ustilago maydis.
403
Concentration tested (mg/mL) 1=10; 2=5; 3=2.5; 4=1.25 and 5=0.70.
404
405
Figure 4. Bacterial growth in different COS mixture concentrations. The growth was
406
expressed as % of the control O.D.600nm after 24 h of incubation at 37 °C. MIC (red
407
line) and EC50 (blue line).
408
Figure 5. Proliferation of HeLa cells in the presence of COS at different
409
concentrations. Changes in the structure and number of HeLa cells were observed
410
under treatment at all concentrations. Cells grown without COS were used as
411
control.
412
Figure 6. Cytotoxic effect of COS on cervical cancer cells. Cells grown without
413
COS were used as control. (*) represents statistically significant differences. The
414
statistical test used for comparison was the student t-test for unpaired samples with
415
Welch correction, with p = 0.048.
416
Figure 7. Purification profile of COS using biogel p-4. A) Elution peaks of COS
417
detected by DNS method. B) TLC of the selected peaks. 1) p1-p2; 2) p3-p5; 3) p6-
418
p8. GlcN=glucosamine; C3-C7= trimer-heptamer.
419 420 421
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Highlights •
Trichoderma harzianum chitinase is a potential system for COS production
•
T. harzianum enzymes produces a COS mixture from monomers to hexamers.
•
The maximum yield of COS was obtained using solid crystal chitosan and fungus spores
•
COS mixture has an effective antimicrobial and anticancer effect
Conflict of Interest Statement
The author declares that the research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.