Process optimization, purification and characterization of a novel acidic, thermostable chitinase from Humicola grisea

Article abstract of DOI:10.1016/j.ijbiomac.2018.05.125

An extracellular acidic and thermostable chitinase (HgChi) from thermophilic Humicola grisea was purified and characterized. Enhancement in chitinase production (Q_p = 2.9662 Ul^?1 h^?1) was achieved through derivation of optimum fermentation conditions via central composite design. H. grisea observed to produce various isoforms of chitinase, among which the major expressed form has molecular mass of about 50 kDa. Purified chitinases exhibited optimal activity at pH 3.0 and 70 °C. Chitinase showed notable stability at increasing temperatures. Half-life of chitinase is 169.06 min at optimum temperature. Chitinase has effectively catalyzed N-acetyl chitobiose (GlcNAc)₂, and N-acetyl chitotriose (GlcNAc)₃ and colloidal chitin. Purified chitinase from H. grisea showed high affinity towards colloidal chitin as evident by its comparatively lower K_m value. Presence of metal ions viz. Mn²⁺, Co²⁺, NH₄ ⁺ and Mg²⁺ significantly increased the chitinase activity. Thin layer chromatography (TLC) analysis revealed the significant hydrolyzing competence of HgChi for colloidal chitin, (GlcNAc)₃ and (GlcNAc)₂ into oligomers and N-acetyl–D-glucosamine (GlcNAc). Thermostable chitinase appeared as potential candidate for efficient conversion of chitin to bioactive oligosaccharides at industrial scale.

Full text of DOI:10.1016/j.ijbiomac.2018.05.125

Accepted Manuscript
Process optimization, purification and characterization of a novel
acidic, thermostable chitinase from Humicola grisea
Manish Kumar, Amandeep Brar, V. Vivekanand, Nidhi Pareek
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doi:10.1016/j.ijbiomac.2018.05.125
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ACCEPTED MANUSCRIPT
Process optimization, purification and characterization of a novel acidic, thermostable
chitinase from Humicola grisea
Manish Kumara, Amandeep Brara, V Vivekanandb and Nidhi Pareeka*
aDepartment of Microbiology, School of Life Sciences, Central University of Rajasthan,
Ajmer-305817, Rajasthan, India
bCentre for Energy and Environment, Malaviya National Institute of Technology, Jaipur
302017, Rajasthan, India
*Corresponding Author
E-mail: nidhipareek@curaj.ac.in; Tel: +91-1463-238651; Fax: +91-1463-238722
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Abstract
An extracellular acidic and thermostable chitinase (HgChi) from thermophilic Humicola
grisea was purified and characterized. Enhancement in chitinase production (Qp = 2.9662 Ul-1
h-1) was achieved through derivation of optimum fermentation conditions via central
composite design. H. grisea observed to produce various isoforms of chitinase, among which
the major expressed form has molecular mass of about 50 kDa. Purified chitinases exhibited
optimal activity at pH 3.0 and 70 0C. Chitinase showed notable stability at increasing
temperatures. Half-life of chitinase is 169.06 min at optimum temperature. Chitinase has
effectively catalyzed N-acetyl chitobiose (GlcNAc)2, and N-acetyl chitotriose (GlcNAc)3 and
colloidal chitin. Purified chitinase from H. grisea showed high affinity towards colloidal
chitin as evident by its comparatively lower Km value. Presence of metal ions viz. Mn2+, Co2+,
+
NH4 and Mg2+ significantly increased the chitinase activity. Thin layer chromatography
(TLC) analysis revealed the significant hydrolyzing competence of HgChi for colloidal
chitin, (GlcNAc)3 and (GlcNAc)2 into oligomers and N-acetyl–D-glucosamine (GlcNAc).
Thermostable chitinase appeared as potential candidate for efficient conversion of chitin to
bioactive oligosaccharides at industrial scale.
Keywords: Chitinase, H. grisea, optimization, purification, thermodynamics, kinetics
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1. Introduction
Chitin, the second most ubiquitous natural biopolymer on earth after cellulose, is a co-
polymer GlcNAc and D-glucosamine (GlcN) subunits linked by β-(1,4) glycosidic
bonds [1]. It is predominantly present in the exoskeletons or cuticles of invertebrates
and in the fungal cell wall. About 1012-1014 tons of chitin is produced annually in the
biosphere with the major contributor being marine ecosystem [2]. Sea food processing
industries are harvesting the marine resources to fulfill the requirements of mankind
chiefly as food, resulting in the generation of huge amounts of chitin-containing waste
in the coastal areas. Despite of its gigantic abundance, utilization of chitinous waste is
still in its primitive stage. The key barrier in the utilization of chitin is its crystallinity
and insolubility [3]. The utilization of chitinous waste could be expanded by its inter-
conversion into derivatives viz. chitosan (CHS), chitooligosaccharides (COS), GlcN
etc., with novel and improved characteristics. The bioconversion of chitin for generation
of high-value commercial products could also provide an economic, sustainable and
environmentally solution to coastal pollution reduction [4].
COS, the major degradation products of chitin are linear co-oligomers of
GlcNAc and GlcN units in varying proportions. A range of bioactivities viz.
antimicrobial, antioxidant, immune cell proliferation effects, gene therapy and tumor
growth inhibition possessed by COS makes their production and application is a
foremost area of research [5, 6]. COS can be produced by physical, chemical and
biological routes, however, enzyme technology involving chitinases is one of the most
promising approach to produce COS with specific degree of polymerization,
acetylation/deacetylation and bioactivities.
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Chitinase (EC 3.2.1.14), member of glycosyl hydrolase family have widely been
employed and explored in the hydrolytic reactions to produce COS. In nature, chitinase
has a broad distribution including bacteria, fungi, nematodes, plants, insects, fish and
human with a vast range of functions including nutrition, morphogenesis, pathogenesis,
parasitism, growth regulation, defense and immunity [7, 8]. Production and application
of microbial chitinase from Aspergillus flavus H5 [9], Trichoderma harzianum P1 [10]
and Penicillium janthinellum [11] has been extensively researched and patented. Recent
advancements in chitin research have paved the way to utilize microbial-derived
chitinase to produce well-defined COS via overcoming recalcitrance of chitin. However,
the development of efficient and effective bioprocess for COS production is in its
infancy due to the scarcity of potent chitinase producing organisms, high production
cost, low activity and stability of available chitinase and partial understanding of its
mode of action to control degree of acetylation/deacetylation and polymerization of
COS [12]. So, it would be pertinent to explore and characterized novel extracellular
chitinase producers, which may produce remarkably higher levels of enzymes.
Moreover, the stability of enzyme at high temperature and pH can amplify their
commercial exploitation as compared to their mesophilic and neutrally stable
counterparts. Additionally, optimization of fermentation conditions could be helpful to
decrease the production cost of enzyme. The optimized media and external factors play
a crucial role in the several folds enhancement of enzyme production [13, 14].
Thermodynamic and kinetic parameters would provide additional information about
factors affecting thermal stability of enzyme along with the suitable substrate for
enhanced COS production.
In this perspective, process optimization for enhanced chitinase production from
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thermophilic H. grisea was carried out employing central composite design (CCD).
Subsequently, the enzyme was purified to homogeneity and kinetics of thermal
inactivation of enzyme was studied by evaluating thermodynamic parameters. Kinetic
parameters were also determined to get an idea about most suitable substrates,
concentration range and catalytic efficiency of enzyme. Further, hydrolyzing efficiency
of chitinase was evaluated using colloidal chitin, (GlcNAc)3 and (GlcNAc)2. The study
would be advantageous to decipher the characteristic features of the chitinase that
materializes its compatibility for bioconversion reactions.
2. Materials and Methods
2.1 Polymers, oligomers and chemicals
Shrimp shell chitin was procured from HiMedia, India. CM-sepharose, DEAE-
sepharose, Calcofluor white M2R and COS with degree of polymerization (2-3) were
obtained from Sigma-Aldrich Co., USA. TLC silica gel sheets were purchased from
Merck, Germany. All other chemicals used were of analytical grade.
2.2 Microorganism
H. grisea ITCC 10,360.16 isolated and characterized in biocatalysis and
bioprocessing laboratory, Department of Microbiology, Central University of Rajasthan,
India from soil sample of Jaisalmer (26° 54′ 41.9796″ N, 70° 55′ 22.5408″ E),
Rajasthan, India was used in the study. [15]. The strain was maintained on potato
dextrose agar (PDA) slants, stored at 4°C and subcultured periodically.
2.3 Chitinase production
Chitinase production by H. grisea was carried out under submerged fermentation
(SF) employing media containing an appropriate ratio of chitin and colloidal chitin as
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carbon source [15]. Briefly, the SF was performed in Erlenmeyer flasks (250 ml) using
50 ml of medium contained (gl ̵
1): chitin, 7.49; colloidal chitin, 4.91; K2HPO4, 0.87;
KH2PO4, 0.68; KCl, 0.19; NH4Cl, 1.0; MgSO4.7H2O, 0.2 and yeast extract, 5.50 and the
pH was adjusted to 6.5. After doing heat sterilization (121 °C, 20 min), the medium was
inoculated with 4 discs (8mm diameter) of freshly grown fungal culture and incubated
(45 °C, 150 rpm, 72 hours). The fermentation broth was centrifuged (8000 g, 10 min)
and the supernatant was used as the enzyme source for further studies.
2.4 Chitinase assay
Chitinase was assayed using colloidal chitin (1%) as a substrate by estimating the
reducing sugar as described by Miller [16]. The assay was carried out by incubating
enzyme and substrate in 1:1 ratio (v/v) at 70 °C for 60 min. Sodium phosphate buffer
(100 mM, pH 6.0) was used for chitinase assay throughout the process optimization and
citrate buffer (100 mM, pH 3.0) was employed for further activity calculations
employing purified chitinase. One unit (U) of chitinase activity was defined as the
amount of enzyme required to release 1µmol of GlcNAc per minute under the standard
assay conditions.
2.5 Optimization of fermentation parameters by central composite design
The optimum fermentation conditions (pH, temperature and inoculum size) for
enhanced chitinase production by H. grisea were derived by employing CCD under
submerged conditions through the statistical software package “Design-Expert version
10”, State-Ease Inc., Minneapolis, USA. The three variables were studied at five coded
levels (-α, -1, 0, +1, +α, where α=2K/4, k is the number of variables) in a set of 20
different experiments performed in triplicates (Table 1). The response (Y) was taken
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from the mean value of chitinase activity (Ul-1). An experimental model was plotted
through the analysis of variance (ANOVA) in order to establish a relation between
independent variables and the observed responses. The relation revealed about the
statistical significance of the model and its behaviour can be explained by the second-
order polynomial equation for a three-factor system (Eq. 1):
3
3
3
Y= β0 + ∑ βiXi + ∑ βiiXi2 + ∑ βijXij
i=1 i=1 ij=1
(1)
Where Y is the predicted response, β0 is offset term, βi are coefficient for the linear
terms, βii are the square terms, βij are the coefficient of the interactive terms. Most
favourable levels of all the three independent variables were determined by Eq. 1 along
with the analysis of the response surface contour plots. Conducting the chitinase
production under the conditions predicted validated the model. Samples were drawn at
specified intervals and the analysis for chitinase production was done as described
earlier.
2.6 Purification of chitinase
The crude extracellular chitinase was subjected to three-step purification process
comprised of concentration, cation and the anion exchange chromatography. The culture
broth was centrifuged at 8000 x g for 10 min at 15 °C. The crude enzyme supernatant
thus obtained was concentrated using activated dialysis membrane (10 kDa molecular
weight cut-off, HiMedia, India) by applying sucrose bed. The concentrated chitinase
obtained from the dialysis was assayed for chitinase activity and protein content.
Further, the concentrated enzyme was loaded on to CM-sepharose (Sigma-Aldrich Co.,
USA) column equilibrated with citrate buffer (100 mM, pH 3.0). Sodium chloride
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gradient (50-500 mM) was employed for the elution of protein (flow rate, 1.0 ml min-1).
Fractions were collected and analyzed for chitinase activity and protein content.
Chitinase active fractions were pooled and further purified using DEAE-sepharose
(Sigma-Aldrich Co., USA) column as mentioned above. The active fractions were
pooled and used for the further studies. Bradford [17] method was used for protein
estimation using bovine serum albumin as a standard and sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described by
Laemmli [18].
2.7 Zymogram analysis
For zymogram analysis, proteins were separated with 10% polyacrylamide native
gel containing 0.1% colloidal chitin and after that the gel was immersed in citrate buffer
(100 mM, pH 3.0) at 60 °C for 180 min followed by staining with 0.01% (w/v)
Calcofluor white M2R [19] and washing with distilled water. Lytic zone was visualized
by placing the gels on a UV-transilluminator.
2.8 Biochemical analysis of the purified chitinase
2.8.1 Determination of optimum pH and stability
The optimum pH for the purified chitinase was detected by analyzing its activity
over a pH range of 2.0-11.0 using the following buffers: 100 mM hydrochloride
acid/potassium chloride buffer (2.0), 100 mM citrate buffer (3.0-5.0), 100 mM
phosphate buffer (6.0-8.0) and 100 mM carbonate-bicarbonate buffer (9.0-11.0). The
stability of enzyme at varying pH was determined by pre-incubating the purified
enzyme in the buffers mentioned above for 24 h at 4 °C and subsequently analyzed the
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residual activity under standard assay conditions. The experiments were performed in
triplicates and their mean value was utilized for activity calculations.
2.8.2 Effect of optimum temperature and stability of chitinase
The optimum temperature was determined by assaying the chitinase activity at
various temperatures (30-90 °C) for 60 min in 100 mM citrate buffer (pH 3.0). The
thermostability of the enzyme was determined by pre-incubating the enzyme at various
temperatures ranging from 70 to 85 °C. Aliquots were withdrawn at various time
intervals, allowed to cool and the enzyme assay procedure described above was
followed.
2.8.3 Thermodynamics of chitinase inactivation
Activation energy (Ea,d) of the purified chitinase was assessed using the slope of
Arrhenius plot. Half-life of the chitinase was estimated according to equation (2). Other
thermodynamics parameters for chitinase activation of denaturation viz. free energy
(ΔGd), enthalpy (ΔHd) and entropy (ΔSd) were obtained by employing equations (3-5).
t1/2 = ln (2)/ kd
(2)
(3)
(4)
(5)
ΔGd = -RT ln (kdh / kb T)
ΔHd = Ea,d- RT
ΔSd = (ΔHd - ΔGd) / T
here, kd is the first-order rate constant (thermal deactivation constant) which
was obtained from the slope of the relative activity (%) vs time at different
temperatures, h is the Planck constant (6.626 × 10-34 Js), R is Gas constant (8.314 J mol-
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1 K-1) , kb is the Boltzmann constant (1.381 × 10-23 J K-1) and T is thermodynamic
temperature (K).
2.8.4 Determination of kinetic parameters
The kinetic parameters of the purified chitinase were determined by measuring
the enzyme activity at different concentrations (0.5-2.5 mg ml-1) of chitin oligomers
(chitobiose, chitotriose) and colloidal chitin. The assay was conducted in triplicates and
the various kinetic constants viz. Vmax, Km, kcat were estimated through the linear
regression plots of Lineweaver and Burk.
2.8.5 Effects of various metal ions and EDTA on chitinase activity
+
Effects of various metal ions ( Na+, K+, NH4 , Ni2+, Cu2+, Ca2+, Hg2+, Cr2+, Fe2+,
Co2+, Ba2+, Mn2+ , Zn2+, Li2+, Mg2+) and EDTA on the chitinase activity was studied by
pre-incubating the purified enzyme in the 100 mM citrate buffer (pH 3.0) with a metal
ion (1 mM). Subsequent to incubation (1 h), residual enzyme activity was measured
under the standard assay conditions. The activity assayed after dialyzed against citrate
buffer (100 mM, pH 3.0) was taken as the control.
2.9
Time-course hydrolytic properties of chitinase from H. grisea
Hydrolytic efficiency of HgChi was analyzed employing TLC using colloidal
chitin, (GlcNAc)3 and (GlcNAc)2 as the substrates. The reaction mixture containing
0.1% (w/v) substrate prepared in 100 mM citrate buffer (pH 3.0) with 0.3 U ml-1 of the
purified chitinase was incubated at 70 °C. Aliquots (100 µl) were withdrawn at various
time intervals and the reaction was stopped by boiling for 10 min, followed by
centrifugation (10000g, 5 min). The samples were concentrated, spotted on to a silica
gel plate (TLC Silica gel 60, Merck, Germany) and developed in a solvent system [20],
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followed by spraying with aniline-diphenylamine reagent [20]. The TLC sheets were
analyzed after heating in an oven for 10 min at 150 °C.
3. Results and Discussion
3.1 Determination of optimum fermentation conditions
Development of an economic and efficient bioprocess for enzyme production relies
on cost-effective production medium and optimum fermentation conditions. The
optimum level of fermentation medium and conditions can be obtained through the
statistical optimization methods viz. response surface methodology (RSM) that allows
study of effects of various factors at a time along with determination of their interactive
effect on production. Effect of three fermentation variables (pH, temperature and
inoculum size) on chitinase production was evaluated by CCD with the help of five
coded levels of three variables (k=3). The CCD design and the corresponding responses
are shown in Table 2. By applying ANOVA on the experimental data, the results of
CCD were put into the following second-order polynomial equation in order to explain
chitinase production as a function of studied variables (Eq. 6).
Y= 374.41 – 40.08 A + 17.71 B – 299.29 C + 1.20 AB + 4.07 AC + 5.88 BC – 4.24A2 -
0.48 B2 + 2.26 C2
(6)
Where, Y represents chitinase production (Ul-1) and A, B and C are coded values of pH,
temperature and inoculum size, respectively.
The very low probability value [(Pmodel F) < 0.0001] of the Fisher’s F test for
the model proved the significance of the model. ANOVA for chitinase production
indicated the ‘F value’ to be 13.86, which illustrated the high significance of the model
in approximating the response surface of the experimental design. Corresponding
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probability of failure value (>F) showed that there were only 0.01 % chances for the
occurrence of this value due to noise. The goodness of fit of the model was examined by
coefficient of determination (R2) and it was found to be 0.9258, which can be used to
explain up to 92.58 % variability of the response. The ‘adequate precision’ value of
14.19 directed an adequate signal and suggested that the model can be used to navigate
the design space. The low value of coefficient of variance (CV = 11.89) indicated that
experiments were conducted with high precision. The ‘‘lack of fit test’’ compares the
residual error to the pure error from replicated design points. The significance of each
coefficient on the response was evaluated by Student’s t-test and Probability of failure
value. Model terms with ‘Prob>[F]’ values < 0.05 were considered significant while
insignificant terms have values > 0.10. According to the model, linear terms of pH and
temperature have noteworthy effect on chitinase production. Additionally, the squared
term of temperature and the interaction of temperature with inoculum size also
significantly affected chitinase production (P-value < 0.05). The uncoded optimum
values calculated from the model equation were 6.49, 47 0C and 5 discs for pH,
temperature and inoculum size, respectively.
Employment of RSM for optimization studies also provides an insight about the
effect of interaction of independent variables on the response. Interaction between
temperature and inoculum size observed to have notable effects on chitinase production
by H. grisea (Fig. 1a, b). It is evident from the response surface plot that the effect of
temperature is more pronounced on chitinase production as compared to inoculum size
(Fig. 1b). Likewise, inoculum is considered as one of the most important factors
affecting enzyme production during fermentation processes. Increase in both the
inoculum size and temperature positively affected chitinase production but up to a
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certain extent. Addition of inoculum beyond a definite range may led to early
exhaustion of rapidly utilizable carbon source in the medium that further resulted into
excessive biomass production and lower levels of enzymes. Also, extremely high
temperature may be detrimental to cell growth and may led to formation of some other
proteins required for survival of cell rather than production of desired molecules i.e.
enzymes. The utilization of statistical means to optimize fermentation conditions had
exploited by many researchers successfully to increase the enzyme titres over the one
factor at a time optimization [21, 22].
Estimating chitinase production using derived conditions did verification of the
model equation. The optimized conditions had resulted into actual maximal chitinase
production of 213.57 ± 6.4 Ul-1 and was observed to be in good agreement with the
theoretically predicted values by the quadratic model (202 Ul-1) (Fig. 2). Optimization
of process parameters had led to 1.24 folds enhancement in chitinase titres.
Lacanicillium muscarium CCFEE-5003 showed similar chitinase production (243 ±
17.3 Ul-1) following employment of optimized substrate concentration and pH
conditions (through RSM) in a bioreactor [23]. Our results are in good agreement with
the study. However, Bacillus licheniformis AT6 (505.26 ± 22.223 Ul-1) [24] and
Pseudomonas fluorescens HN1205 (1030 Ul-1) [25] exhibited relatively enhanced
chitinase production titres following optimization.
3.2 Chitinase purification and molecular mass determination
Extracellular HgChi (50 kDa) was purified from the crude culture supernatant by
employing a three-step purification process consisting of concentration followed by
cation and anion exchange chromatography (Table 3, Fig. 3). Patil et al. [26] also
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reported production of extracellular chitinase (40 °C, 120 rpm and 96 h) from
thermophilic Penicillum ochrochloron and purified through DEAE-cellulose anion
exchange chromatography followed by molecular mass determination (64 kDa) through
SDS-PAGE. The molecular mass of the purified fungal chitinase lies in the range of 30-
150 kDa [27-30].
3.3 Effects of pH on chitinase activity and stability
Purified chitinase exhibited maximum activity at pH 3.0 (Fig. 4a). A sharp decline
in chitinase activity was observed at all other pH ranges studied. Enzyme’s pH stability
analysis had shown that the enzyme retained about 90% of its activity at pH 3.0
following 24 h of incubation (Fig. 4b). Moreover, at higher pH 9.0-11.0 enzyme was
able to retain more than 80% of activity. Mander et al. [31] had also reported high pH
(5.0-13.0) stability of the purified chitinase obtained from Streptomyces anulatus after
the incubation for 48 h at 4 °C. Chitinase exhibiting high acid or base tolerating
capacity has also reported by several researchers [32, 33]. The higher stability of
chitinase from H. grisea at low pH advocates its applicability as a suitable bio-catalytic
solution for large scale applications for bioconversion of chitin waste to the value-added
COS.
3.4 Thermal kinetics of chitinase
The purified chitinase showed maximum activity at 70 0C. Additional incubation at
higher temperatures led to reduction in enzyme activity. Time-dependent thermal
inactivation kinetics of enzyme was studied by incubating it at various temperatures
(70-85 0C) to get an idea about thermal stability of enzyme at higher temperatures. It
was observed that the enzyme retained notable activity at relatively higher temperatures.
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The HgChi temperature optima (70 °C) was comparable to the patents related to the
thermostable chitinase from recombinant Vibrio parahemolyticus strain (45 °C) [34]
and Brevibacillus later osporus LAK 1210 (70 °C) [35].
Thermodynamic parameters viz. half-life, free energy, enthalpy and entropy predicts
about the stability of enzyme against thermal denaturation. Half-life of the purified
chitinase (i.e. enzyme thermostability indicator) at its optimum temperature was
169.060 min which was observed to reduce following increment in incubation
temperature (Table 4). First-order rate constants (kd) for thermal denaturation of HgChi
at various temperatures were determined from the slopes of the first order plot (%
relative activity vs time). In order to achieve complete denaturation of enzyme a least
amount of energy is compulsory which is known as activation energy of denaturation
(Ea,d). The activation energy of denaturation for HgChi was estimated to be 108.91 kJ
mol-1 (Fig. 5). At optimum temperature for HgChi, the values of ΔGd, ΔHd, and ΔSd
were 100.079 kJ mol-1, 111.765 kJ mol-1 and 34.069 J mol-1 K-1, respectively (Table 4).
The positive values of ΔSd suggested the deactivation of chitinase following disruption
of non-covalent bonds at high temperatures. There is no significant correlation was
observed between ΔGd and temperature as there is no considerable decrease in ΔGd
value with increasing temperature (Table 4). Similar relation was also observed for the
enthalpy of activation for the thermal inactivation of HgChi with increasing
temperature. The results are in agreement with the studies performed to investigate the
thermal denaturation of chitinase [36], chitin deacetylase [37] and serine protease [38].
3.5 Kinetic parameters of chitinase
Kinetic parameters viz. Vmax, Km, kcat and kcat / Km of purified chitinase were
determined through Lineweaver-Burk plot using various substrates i.e. colloidal chitin,
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(GlcNAc)2 and (GlcNAc)3. The purified chitinase showed significant affinity towards
the substrates studied. (GlcNAc)2 and (GlcNAc)3 appeared to be preferable substrates
by enzyme for catalysis. The affinity of purified chitinase towards (GlcNAc)2 and
(GlcNAc)3 was almost similar, as evident from their Km values (0.30 mg ml-1 for
(GlcNAc)2 and 0.33 mg ml-1 for (GlcNAc)3). The enzyme also exhibited similar Vmax
values for the substrates (1.29 µmol min-1 for (GlcNAc)2 and 1.25 µmol min-1 for
(GlcNAc)3) (Table 5). HgChi exhibited highest affinity towards colloidal chitin as
suggested by its lower Km value (0.11 mg ml-1) (Table 5). However, the relatively less
Vmax value (0.84 µmol min-1) for colloidal chitin could be attributed to its more
crystalline nature as compared to the oligomers that restricts the substrate accessibility.
The values of Michaelis-Menten constant and specificity constant signifies substantial
catalytic activity of the purified chitinase towards (GlcNAc)2 and a decrease in the
enzyme affinity was observed with increase in the degree of polymerization. Prasad and
Palanivelu [39] studied kinetics of a 42 kDa recombinant chitinase from thermophilic
fungus Thermomyces lanuginosus and reported Km and Vmax values of 0.147 mM and
814 mM min-1 mg-1 employing colloidal chitin as the substrate. HgChi appeared to be
posses better affinity towards colloidal chitin with lower Km (0.11 mg ml-1) and higher
kcat (14 s-1) compared to the chitinase from Penicillium ochrochloron (Km, 1.3 mg ml-1;
kcat , 2.37 s-1) [40].
3.6 Effects of metal ions on enzyme activity
Metals can play role as co-factors during enzyme catalysis and may enhance activity
of certain enzymes. Effect of various metal ions and EDTA on chitinase activity was
assayed to detect the metal ion requirement for catalysis. Among the different metal
+
ions studied Mn2+, Co2+, NH4 and Mg2+ appeared as activators of chitinase activity,
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however, the chitinase activity was inhibited by Hg2+, Ca2+, Cu2+, K+ and EDTA at 1
mM concentrations (Fig. 6). Among the activators, Mn2+ and Co2+ were significantly
enhancing the chitinase activity up to 173% and 144% respectively. Ni2+, Na+ and Li2+
had no considerable effect on the chitinase activity. In case of inhibitory metal ions,
Ca2+ and Hg2+ were appeared to be the significant inhibitors of chitinase activity,
whereas, Fe2+ and Cr2+ were reported to moderately inhibit chitinase activity. Chitinase
activity inhibition by EDTA suggested that the enzyme is a metallo-protein. Chitinase
activity was observed to be highly affected by Hg2+, Mn2+, Mg2+, Ca2+ and Co2+. Yang
et al. [40] had reported enhancement in chitinase activity by Ca2+ and Ni2+ ions,
however in the present study Ca2+ has appeared as inhibitor of enzyme activity and Ni2+
had no noteworthy effect on the chitinase activity. Fe2+, K+ and Hg2+ were reported to
be inhibitors of chitinase activity whereas; Mg2+ was reported to be activator of
chitinase [26, 41]. The results from the present study showed good agreement with the
previous research findings.
3.7 Hydrolytic properties of H. grisea chitinase
The hydrolytic efficiency of HgChi was investigated on colloidal chitin, (GlcNAc)3
and (GlcNAc)2. The hydrolyzing efficiency of HgChi to produce COS was evaluated by
analyzing the hydrolytic products by TLC using (GlcNAc)3 , (GlcNAc)2 and GlcNAc
(Sigma-Aldrich, USA) as standards. The HgChi was able to efficiently hydrolyzed
colloidal chitin to generate (GlcNAc)3, (GlcNAc)2 and GlcNAc following incubation at
70 0C (30-60 min) (Fig. 7a). Further increase in incubation period (i.e. 60-300 min) had
resulted into augmentation of GlcNAc concentration due to the hydrolysis of (GlcNAc)3
and (GlcNAc)2. The HgChi was also capable of hydrolyzing (GlcNAc)3 into (GlcNAc)2
and GlcNAc following incubation for 5- 30 min (Fig. 7b). Additional elongation in
17

ACCEPTED MANUSCRIPT
hydrolysis time (60-120 min) led to complete conversion of (GlcNAc)3 into GlcNAc.
HgChi also showed to notably hydrolyze (GlcNAc)2 (Fig. 7c). Chitinase illustrated to
completely hydrolyze (GlcNAc)2 into (GlcNAc)2 and GlcNAc within 3 min of
incubation and complete conversion into GlcNAc within 10 min of incubation time. The
results from TLC analysis could be used to deduce preliminary conclusions about mode
of action of HgChi. TLC analysis suggested that HgChi is an endo-acting chitinase as it
showed randon cleavage pattern and produce a mixture of (GlcNAc)3, (GlcNAc)2 and
GlcNAc following hydrolysis. HgChi showed better hydrolyzing capacity towards
colloidal chitin as compared to the extracellular chitinase from Lecanicillium lecanii,
that reported to convert colloidal chitin into GlcNAc following 96 h of incubation at 40
°C [29]. The remarkable hydrolytic potential of HgChi could be attributed to its
thermophilic nature that contributed in enhanced reaction rates. The hydrolyzing ability
of HgChi towards colloidal chitin is comparable to the hydrolyzing efficiency of the
recombinant chitinase developed from thermophilic Themoascus aurantiacus and
Chaetomium thermophilum [42]. Chitinase from T. aurantiacus and C. thermophilum
reported to hydrolyze colloidal chitin to produce (GlcNAc)2 and GlcNAc following
incubation for 30 min at 50 °C [42]. Yang et al. [30] has shown that acidic exochitinase
from Rhizomucor miehei can effectively hydrolyze colloidal chitin, (GlcNAc)3 and
(GlcNAc)2 into various chito-oligomers and monomers.
4 Conclusions
The present study concluded that the optimization of process parameters through RSM
can enhance chitinase production. The chitinase produced by H. grisea can effectively
hydrolyze chitin to produce oligosaccharides. Optimum activity of enzyme at relatively
lower pH (3.0) and higher temperature (70 °C) along with its stability over a broad
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range of temperature advocates its wide applicability from industrial point of view.
High catalytic efficiency and specificity of HgChi supported its high industrial
effectiveness and competence. Further enzyme structural studies would help to reveal its
conformational stability and complexity of denaturation. Additionally, site-directed
mutagenesis could be employed to enhance the activity and stability of chitinase at
extreme operating conditions and also to deduce the structure-function relationships.
Acknowledgements
The authors would like to thank Department of Biotechnology (No. BT/Bio-
CARe/03/9840/2013-14) and Department of Science and Technology (No.
DST/INSPIRE/04/2014/002644), Government of India to support this work financially.
Authors also gratefully acknowledged Central University of Rajasthan, Rajasthan, India
for providing the necessary facility to carry out this work.
Conflict of interest
The authors declare no conflicts of interest.
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Table 1. Concentration ranges of independent variables used in central composite
design
Levels
Variable
Component
pH
Range studied
5.00-7.00
-α
4.31
-1
5.00
0
+1
7.00
+α
A
B
6.00
7.68
40.00-50.00
36.59 40.00 45.00 50.00 53.41
Temperature (0C)
C
3.00-5.00
2.32
3.00
4.00
5.00
5.68
Inoculum size
(No. of discs)
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Table 2. Experimental design matrix for optimization of chitinase production using
central composite design
Run
Independent variables (actual values)
Chitinase (U l-1)
Inoculum size
Temperature (0C) (No. of discs)
pH
7
Observed
Predicted
173.22
132.62
125.72
143.21
132.62
92.057
132.62
145.37
152.30
52.41
1
2
50
45
5
4
165.23
133.63
122.19
149.46
128.01
68.74
6
3
6
45
2.32
3
4
5
40
5
6
45
4
6
5
40
5
7
6
45
4
135.23
141.78
158.30
58.47
8
6
53.40
45
4
9
6
5.68
4
10
11
12
13
14
15
16
17
18
19
20
6
36.59
50
5
5
200.43
169.80
112.39
136.78
73.93
194.12
155.17
127.64
132.62
86.10
4.32
5
45
4
50
3
6
45
4
7.68
7
45
4
50
3
112.03
73.93
90.45
7
40
3
81.97
6
45
4
131.21
130.45
60.62
132.62
132.62
47.10
6
45
4
7
40
5
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Table 3. Purification profile of chitinase from H. grisea
Purification
step
Volume
(ml)
Total
activity (U)
Total
protein
(mg)
Specific
activity
(U mg-1)
Yield (%) Purification
fold
Culture
50
11.72
4.86
2.41
100
1
supernatant
Dialysis
10
14
5.26
3.12
1.91
2.75
0.18
44.88
26.62
1.14
0.07
CM-
17.31
sepharose
DEAE-
20
2
0.22
9.09
17.06
3.77
sepharose
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Table 4. Thermodynamic parameters of chitinase from H. grisea
T (K)
t1/2
(min)
ΔGd
ΔHd
ΔSd
(kJ mol-1)
(kJ mol-1)
(J mol-1 K-1)
343
348
353
358
169.060
93.668
60.802
32.850
100.079
99.871
100.080
99.709
111.765
111.806
111.848
111.889
34.069
34.296
33.337
34.030
29