Kannan Natarajan* and V. Ramachandra murty
Department of Biotechnology, Manipal Institute of technology, Manipal-576104, Karnataka, India.
Chitin, a h-1,4-linked homopolymer of N-acetylglucosamine
is the second most abundant natural renewable polysaccharide and mostly is
present in fungi, algae, insects and marine invertrebrates. Chitinase enzyme
(EC 18.104.22.168) has capable of catalyzing the hydrolysis of chitin to its monomer
N-acetyl-D-glucosamine from variety of sources, such as bacteria, fungi, yeast
and plants (Patil et al., 2000). Chitinase enzyme is most promptly used in the
biological research as a controlling agent for the generation of fungal
protoplasts due to its degrading nature of fungal cell wall. Chitinase is a
hydrolytic enzyme; this property makes them as an alternative environmental
biological control agent (Ordentlich et al., 1988). And also chitinase find
widespread applications in the field of agriculture, medicine, biochemical
processing engineering, waste management, pesticide control and cell wall
Several microorganisms, including bacteria such as Bacillus
lichiniformis, Bacillus pabuli, Bacillus thuringiensis, Serratia
marcescens, Nocardia orientails ,Vibrio alginolyticus (Mabuchi et al., 2000; Someya et al., 2001;
Wen et al., 2002 and Huang et al., 2005) and many species of fungi such
as: Myrothecium verucaria , Stachybotrys elengans Streptomyces cinereorube, Streptomyces
lydicuis, Trichoderma harzianum, Trichoderma viride, Verticillium
lecanii (Mathivanan et al., 1998
and Viterbo et al., 2001) have a chitinase producing ability. Chitinase
activity in plant and human serum has also been described recently (Mathivanan
et al., 1998). Novel bacteria strains are described which are created by the
introduction of DNA encoding for the production of chitinase.
In this present study a well known chitinolytic bacterial
strain of Serratia marcescens has
chosen to the production of chitinase, to ensure the maximum production of
chitinase. The different medium culture was taken initially to find out the initial
range of production of chitinase. And significance of the medium composition
and other physicochemical parameters were optimized by the classical approach
to screen the most prompt factors affecting the enzyme production by the
chitinolytic bacterial strain.
maintenance and inoculum preparation
The chitinolytic bacterium was obtained from our
laboratory, in Manipal Institute of technology, Manipal University, India. The
organism was cultivated on LB medium consisting of (g/l) Agar 15; tryptone 10;
sodium chloride 10 and yeast extract 5.0. The pH of the medium was adjusted to
7.2 using 1 M NaOH or 1 N HCl and sterilized by autoclaving at 121ºC for 15
min. The production medium was inoculated with 5% (v/v) of seed culture in the
mid exponential phase at 30 h (Fig. 1). The flasks were incubated in an orbital
shaker at 120 rpm and 30°C for the fermentation period of 60 h. Aliquot of sample
from the fermentation broth was withdrawn at 6 h interval without much change
in the culture volume to maintain constant oxygen transfer. The cells were
separated from the medium by centrifugation at 10,000 rpm for 15 minutes. The
clarified supernatant was used for the analysis of chitinase activity.
The bacterial cell growth was determined by measuring the optical density at
wavelength of 600 nm (Double beam UV Visible spectrophotometer).The biomass
concentration was determined with a calibration curve made from the relationship
between optical density at 600 nm and dry cell weight (Fig 1).
Extracellular chitinolytic activity in the broth may
calculate using chitin as the substrate. One milliliter of the supernatant
broth was mixed with 1ml of 1% chitin in 0.05M phosphate buffer pH 7.0. The assay
mixture was incubated at 40ºC and the end products of the reaction were
analyzed using the DNS method. One unit of chitinase activity is defined as the
amount of enzyme required to release 1 micro mol of GlcNAc in 1 min under the
above mentioned conditions.
Effect of different media compositions
Three different media namely M1, M2 and M3 amended with 1% colloidal chitin
were used to determine the growth of Serratia marcescens and chitinase
production. 5% (v/v) of seed culture in the mid exponential phase at 30 h was
inoculated with 100 ml of each medium and incubated at 100 rpm in a rotary shaker
at room temperature. After two days of incubation, the cultures were harvested,
centrifuged at 10000 rpm for 15 min and the supernatant was used for chitinase
of physicochemical parameters
The optimized medium culture involves further
optimization of fermentative physicochemical parameters, such pH, temperature
and agitation speed.
RESULTS AND DISCUSSION
A amount of literature gives
the detailed information about the microbial production of chitinase and inspection
about the effect of important physicochemical and environmental factors, exclusively
culture condition, culture composition, substrate concentration, pH,
temperature, agitation speed, incubation period, inoculation volume, aeration
rate, etc.,had significant effect on
production of chitinase and biomass.
In biochemical investigation
of enzyme production mainly depends on the culture compositions, it acts the
important role for the growth as
well as metabolites production by microorganisms. The kinetic profile of
cell mass and chitinase production by S.
marcescens grown in different media was illustrated in Fig. 2. The cell
growth and chitinase production was high in the order M3>M2>M1 as shown
in Fig. 2. The chitinase production by S.
marcescens was found to be growth associated in all the fermentation runs
conducted, as the chitinase production increased with the cell mass. Maximum cell
mass was obtained in the media M3 which contains chitin as the major carbon
source. Among the three medium compositions, M3 medium was found to be a best
chitinase producing culture medium than other media M1 and M2 compositions.
Medium M3 was chosen to further optimizing the physicochemical and
environmental parameters. The composition of M3 medium contains the chitin as a
sole carbon source combined with other nitrogen and mineral sources.The concentration of chitin is plays a major
role, important factor to induce the chitinase production in microbial
production. The different concentration of chitin enhances the production of
chitinase, at 0.5%
concentrations; chitin significantly enhanced the chitinase activity. Mathivanan
et al., 1998 reported that the same concentration of chitin has the ability to
produce chitinase in Fusarium chlamydosporum. The addition of chitin
concentration above 0.5% also induced the maximum chitinase production in Bacillus
sp. NCTV2 (Wen et al., 2002), Alternaria alternate (Sharaf, 2005) and Trichoderma harzianum (Sandhyal
et al., 2005).
various carbon sources on chitinase production
For the growth of microorganism carbon source is the very essential with other
nutrient sources, etc. The M3 medium was taken to further studies to evaluate
the effect of carbon and nitrogen sources. Various carbon sources included chitin,
arabinose, cellulose, glucose, galactose, maltose, fructose, sucrose, lactose
and starch were examined at the concentration of 5 (g/L). The samples were taken
at different time interval for the analysis of biomass and chitinase activity.
The above mentioned carbon sources produces chitinase in very low, except chitin.
Then the experiments were conducted in the presence and absence of chitin. The
experimental result implies that presents of chitin activity increases the production
of chitinase, but the absence of chitin doesnot improve the final reaction production.
The exception of carbon sources like, cellulose and arabinose were shown the
increased activity than the other carbon sources used.These experimental results
were conformity with Vaidya, et al., 2001 reported that the production of chitinase
with different carbon sources, no detectable activity in final production with
some exceptional carbon sources. Gupta et al., 1995 reported that in Streptomyces
viridificans, arabinose with chitin doubled the production of chitinase
but arabinose alone failed to induce the chitinase. The carbon is very essential
for the growth of the organism. The increased order of chitinase production
by chitinolytic bacterial strain Serratia marcescens using different
carbon sources was chitin>cellulose>arabinose>glucose>starch>sucrose>lactose>maltose
(Fig 3). From the results of different carbon sources, found that chitin alone
act as inducer and best carbon source to improve the chitinase production. Maximum
production of chitinase obtained in M. verrucaria was observed with chitin
as a carbon source and no detectable activity was seen in the culture grown
with lactose, maltose, sucrose, chitosan, starch and cellulose (Vyas et al.,
various nitrogen sources on chitinase production
Nitrogen sources are very important for the microbial
growth and to maximize the final reaction product next to carbon sources. For
nitrogen sources the medium was supplemented with peptone, yeast extract,
potassium dihydrogen phosphate, ammonium sulphate and ammonium chloride in
equivalent concentrations to the production medium.
The order of chitinase production using various nitrogen sources follows peptone>
yeast extract> ammonium sulphate> ammonium chloride> potassium dihydrogen
phosphate (Fig 4). Among the various nitrogen sources involved in reaction,
the experimental results were shown pepote and yeast extract has the significant
increasing order than other sources added to the medium. The optimal value of
nitrogen source of the media for chitinase production was 1 g/L. Addition of
yeast extract to the medium has been reported to enhance enzyme production in
Serratia marcescens (Monreal and Reese, 1969) and Aspergillus carneus
(Sherief et al., 1991). The ammonium sulphate and ammonium chloride also gave
the response next to the above mentioned nitrogen sources. Vyas and Deshpande,
1991 reported that in Myrothecium verrucaria, 0.14% ammonium sulphate
and 0.01±0.05% urea increased the chitinase production up to fourfold, while
sodium nitrate favored chitinase production in Stachybotrys elegans (Tweddell
et al., 1994).
The analyzed various carbon and nitrogen sources gives the
neat information about to optimize the culture medium compositions, for further
studies of optimizing the culture environment to maximize the chitinase
production. The optimized medium composition obtained with the chitinase activity
of 24.2 units/mL.
of physicochemical parameters by classical approach
Effect of pH
on chitinase production
Normally the action of initial pH of fermentation medium influences the final
yield of chitinase. Any change in pH affects the protein structure and a decline
in enzyme in activation or its instability. The effect of different levels of
pH was studied to evaluate the final production of chitinase from the nature
of product profile. While in the fermentation aeration rate, agitation rate
and temperature were kept in the constant levels, the pH levels varied from
3 to 10.The culture broth was grown to exponential growth phases and inoculated
on a separate culture medium containing optimized culture compositions. Fig
5 shows the fermentative production of chitinase activity with different pH
levels. The cell growth and chitinase activity were measured in different pH
levels, the maximum chitinase activity of 43.61 units/mL occurred at pH 8. Monreal
and Reese, 1969 and Bergey’s Manual of systemic bacteriology gives the idea
in general S. marcescens had a tendency to be more acidic than pH 7.5.The
pH for optimal production of chitinase using Serratia marcescens was close to
pH 8 reported by Moneal and Reese, 1969. This experimental results leads to
increased stability at high pH with following order 8>7>9>6>5>10>4>3
(Fig 5). The process with pH might force it to change its metabolic pathway
and cause a decrease in the chitinase activity during the cultivation.
temperature on chitinase production
The rate of the reaction increases as the
temperature is raised. The enzyme reaction activity gets increase to two fold
higher with increase of temperature. In the case of enzymatic reactions,
this is complicated by the fact that many enzymes are adversely affected by
high temperatures. As shown in Fig 6, the chitinase enzyme activity increases
with temperature to a maximum level, then abruptly declines with further
increase of temperature. Because most enzymes rapidly become denatured at
temperatures above 40°C, widely the enzyme determinations are carried out
somewhat below that temperature.
The optimized medium was selected to study the effect of temperature on chitinase
production by changing the temperature and kept all other fermentation conditions
were constant. The optimum growth temperature for chitinase production by S.
marcescens was found to be in the range of 30°C (Fig. 6). The activity achieved
at 20°C was lower than the optimum temperature. The enzyme activity decreased
as the temperature increased above 30°C and even at 35°C a 70% decrease in chitinolytic
activity was observed.
Effect of agitation speed on chitinase activity
The optimized culture compositions were taken for the further reaction carried
out to evaluate the optimal shaking speed on chitinase production by subjective
the different agitation speed, with the optimized culture pH and temperature.
The optimum agitation speed for the maximum chitinase activity was found to
be 250 rpm (Fig. 7) upon varying the agitation speed (0- 350 rpm). Agitator
speed of 250 rpm was found to be most suitable for cell growth as well as for
chitinase production. Chitinase yield decreased rapidly at higher agitator speed,
while decrease in cell yield at higher agitator speed was not rapid. Probably,
mass transfer limitation was predominant in the fermentation process at lower
agitator speed. Higher agitator speed appears to reduce chitinase production.
The increase in agitation rate beyond 250 rpm resulted in a drastic fall in
specific enzyme activity. The agitation speed below 150 rpm resulted in inadequate
mixing of the broth towards the later stages of growth. The reaction against
agitation speed indicates that oxygen adjustment is required for the maximum
production of chitinase enzyme (Mathivanan et al., 1998).
From these studies it has been found
that the composition of medium plays a crucial role in the production of
fermented products and optimized levels of carbon and nitrogen sources from the
large number of experimental runs were determined. The chitinase fermentation
process utilizing chitin as the sole carbon source was investigated in a
submerged fermentation process.
The best physicochemical
parameter conditions for maximum production of chitinase
Agitation speed 250
rpm with the combination of optimized medium
compositions contains (g/l): chitin, 5.0; peptone, 1.0; (NH4)2SO4,
1.0; MgSO4·7H2O, 0.3; KH2PO4, 1.36.
Fungal species are most potent producer of this enzyme but the production time
is larger and the initial cost also very high, but in case of bacterial strain
the time consuming for production if comparatively very low than the fungal
species also the range of initial cost also differs. The cultivation time required
for bacterial culture is low and easily can grow and produce huge amount of
enzyme.Further works on optimization of process parameters using statistical
designs, scale up process using fermenter, purification of enzyme by suitable
methods like ATPs, RMs, characterization and regulation of the enzyme synthesis
is in progress.
The authors are thankful to Department of Biotechnology, Manipal Institute
of Technology, Manipal University, Manipal, India for continuous encouragement
and problem solving assistance.
R., Saxena, R. K., Chaturvedi, P., and J. S. Virdi. 1995. Chitinase production
by Streptomyces viridificans: its potential in fungal cell wall lysis. Journal of Appied Bacteriology 78:
C.J., Wang, T. K., Chung, S. C., and C. Y. Chen. 2005. Identification of an
antifungal chitinase from a potential biocontrol agent, Bacillus cereus. Journal
of Biochemistry and Molecular Biological Science 38: 82-88.
Mabuchi, N., Hashizume, I., and Y.
Araki. 2000. Characterization of chitinases exerted by Bacillus cereus CH. Canadian
Journal of Microbiology 46: 370-375.
N., Kabilan, V., and K. Murugesan. 1998. Purification, characterization and
anti-fungal activity from Fusarium chlamydosporum, a mycoparasite to
groundnut rust, Puccinia arachidis. Canadian
Journal of Microbiology 44: 646-651.
J., and E.T. Reese. 1969. The chitinase of serratia
marcescens. Canadian Journal of
Microbiology 15: 689-696.
A., Elad, Y., and I. Chet. 1988. The role of chitinase of Serratia marcescens
in biocontrol of Sclerotium rolfsii. Phytopathology 78: 84-88.
R. S., Ghomade, V., and M. V. Deshpande. 2000. Chitinolytic enzymes: an
exploration. Enzyme Microbial Technology
C., Binod, P., Nampoothiri, K. M., Szakacs, G., and A. Pandey. 2005. Microbial
synthesis of chitinase in solid cultures and its potential as a biocontrol
agent against phytopathogenic fungus Colletotrichum gloeosporioides. Applied Biochemistry and Biotechnology.
E. F. 2005. A potent chitinolytic activity of Alternaria alternate isolated
from Egyptian black sand. Polish Journal
of Microbiology. 54: 145-51.
A. A., El-Sawah, M. M. A., and M. A. Abd El-Naby. 1991. Some properties of
chitinase produced by a potent Aspergillus carneus strain. Applied Microbiology and Biotechnology 35: 228-280.
N., Nakajima, M., Hirayae, K., Hibi, T., and K. Akutsu. 2001. Synergistic
antifungal activity of chitinolytic enzymes and prodigiosin produced by the
biocontrol bacterium Serratia marcescens strain B2 against the gray mold
pathogen, Botrytis cinerea.
Journal ofGeneral Plant Pathology 67: 312-317.
Tim, W., and P. O. Colin. 2002. Comparison of various
plant residues as phosphate rock amendment on Savanna soils of West Africa.
Journal of Agricultural Sciences 12: 321-332.
R. J., Jabaji-Hare, S. H., and P. M. Charest. 1994. Production of chitinases
and b-1,3-glucanases by Stachybotrys elegans, a mycoparasite of Rhizoctonia solani. Applied and Environmental Microbiology 60: 489-495.
Vaidya, R. J., Shah, I. M., Vyas, P.
R., and H. S. Chhatpar. 2001. Production of chitinase and its optimization from
a novel isolate Alcaligenes xylosoxydans: potential antifungal
biocontrol. World Journal of Microbiology and Biotechnology 17:
A., Haran, S., Friesem, D., Ramot, O., and I. Chet. 2001. Antifungal activity
of a novel endochitinase gene (chit36) from Trichoderma harzianum Rifai. TM. FEMS Microbiology Letters.
P., and M. V. Despande. 1991. Enzymatic hydrolysis of chitin by Myrothecium verrucaria chitinase complex
and its utilization to produce SCP. Journal
of General Applied Microbiology 37 (3): 267-275.
C., Tseng, M. C. S., Cheng, C. Y., and Y. K. Li. 2002. Purification,
characterization and cloning of a chitinases from Bacillus sp. NCTU2. Biotechnology and Applied Biochemistry 35: