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Electronic Journal of Biotechnology
Universidad Católica de Valparaíso
ISSN: 0717-3458
Vol. 8, Num. 2, 2005, pp. 134-145
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Electronic Journal of Biotechnology, Vol. 8, No.2, August 15, 2005, pg.
134-145
RESEARCH ARTICLE
Chitinase from Enterobacter sp. NRG4: Its purification,
characterization and reaction pattern
Neetu Dahiya1, Rupinder Tewari2, Ram P. Tiwari3, Gurinder Singh
Hoondal*4
1Department of Biotechnology,
Panjab University,
Chandigarh,
PIN-160014, India,
Tel: 91 172 2534150,
Fax: 91 172 2541409,
E-mail:ineetudahiya@yahoo.com
2Department of Biotechnology,
Panjab University,
Chandigarh,
PIN-160014, India,
Tel: 91 172 2534180,
Fax: 91 172 2541409,
E-mail:rooptt1@glide.net.in
3Department of Microbiology,
Panjab University,
Chandigarh,
PIN-160014, India,
Tel: 91 172 2541770,
Fax: 91 1722541409,
E-mail: rptiwari@rediffmail.com
4Department of Microbiology,
Panjab University,
Chandigarh,
PIN-160014, India,
Tel: 91 172 2541770,
Fax: 91 172 2541409,
E-mail:gshoondal@rediffmail.com
*Corresponding author
Financial support: Senior Research Fellowship from Council of Scientific
and Industrial Research (CSIR), Government of India for Neetu Dahiya.
Received December 15, 2004 /
Accepted April 27, 2005
Code Number: ej05016
Abstract
Enterobacter sp. NRG4 was shown to excrete
chitinase into the culture supernatant when cultivated in medium containing
chitin. A 60 kDa extracellular chitinase was purified to homogeneity and
characterized. The enzyme hydrolyzed swollen chitin, colloidal chitin, regenerated
chitin and glycol chitin but did not hydrolyze chitosan. The chitinase exhibited
Km and Vmax values of 1.43 mg ml-1 and 83.33 µM µg-1 h-1 for
swollen chitin, 1.41 mg ml-1 and 74.07 µM µg-1 h-1 for
colloidal chitin, 1.8 mg ml-1 and 40 µM µg-1 h-1 for
regenerated chitin and 2.0 mg ml-1 and 33.33 µM µg-1 h-1 for
glycol chitin, respectively. The optimal temperature and pH for activity
were 45ºC and pH 5.5, respectively.
Mg2+,
K+ and Ca2+ stimulated chitinase activity by 13, 16
and 18%, respectively whereas Cu2+, Co2+, Ag+ and
Hg2+ inhibited chitinase activity by 9.7, 15, 22 and 72.2%, respectively
at 1 mM concentration. N-bromosuccinamide
(NBS) at 1 mM and iodoacetamide at 10 mM concentration completely inhibited
the enzyme activity. Dithiobisnitrobenzoic acid (DTNB) at 10 mM concentration inhibited chitinase
activity by 97.2%. Chitin was hydrolyzed to chitobiose and N-acetyl D-glucosamine
when incubated with the purified enzyme. The hydrolysis pattern of the purified
enzyme indicated that the chitinase was an endochitinase.
Keywords: chemical modification, chitinase, Enterobacter sp.
NRG4, purification, substrate binding.
Chitin is composed of repeating N-acetyl D-glucosamine residues
and is a component of crustacean exoskeleton, diatoms, fungal cell walls, and
squid pens. Chitin is a versatile and promising biopolymer with numerous industrial,
medical and commercial uses. However, it is difficult to purify and modify
chemically. Hence identification of chitin modifying enzymes and elucidation
of their activities could facilitate the efficient production of specific chitin
products. The biodegradation of chitin requires the synergistic action of several
hydrolytic enzymes for efficient and complete breakdown. The combined action
of endochitinases (EC 3.2.1.14) and exochitinases [(chitobiosidases and β-N-acetyl
hexosaminidase (EC 3.2.1.82)] results in the degradation of chitin polymer
into the soluble N-acetyl D-glucosamine (Gkargkas et al. 2004).
Chitinases are produced by different micro-organisms which generally present
a wide multiplicity of enzymes that are mainly extracellular. They have received
increased attention due to their wide range of biotechnological applications,
especially in the production of chito-oligosaccharides and N-acetyl D-glucosamine
(Pichyangkura et al. 2002), biocontrol of pathogenic fungi
(Chernin et al. 1997; Mathivanan et al. 1998),
preparation of sphaeroplasts and protoplasts from yeast and fungal species
(Mizuno et al. 1997; Balasubramanium et al.
2003) and bioconversion of chitin waste to single cell protein (Vyas
and Deshpande, 1991).
In the present investigation we report an endochitinase that
was purified and characterized from a newly isolated Enterobacter sp.
NRG4.
Materials and Methods
Chemicals and substrates
Flake chitin was obtained from Hi-Media, India.
Swollen chitin was prepared by the method of Monreal and Reese
(1969), colloidal chitin by the method of Jeuniaux (1966),
regenerated chitin by the method of Molano et al. (1977) and
glycol chitin by the method of Yamada and Imoto (1981). N-acetyl
D-glucosamine was obtained from Fluka and chitobiose from Sigma,
Co. All other reagents used were of analytical grade.
Micro-organism and culture
conditions
Enterobacter sp. NRG4 isolated from degraded stalk
of mushroom was selected as a potent chitinase producer (Dahiya
et al. 2005). The culture medium was composed of 1.0% swollen chitin, 0.5%
peptone, 0.5% yeast extract, 0.1% KH2PO4 and 0.01% MgSO4.7H2O
(pH 8.0). The micro-organism was cultivated at 30ºC for
72 hrs with agitation at 150 rpm.
Enzyme and protein
assay
The assay mixture contained 1 ml swollen chitin and 0.5 ml
enzyme solution. After incubation at 45ºC for
15 min, it was centrifuged at 5000 x g for 10 min. The amount of N-acetyl D-glucosamine
released in the supernatant was determined by the method of Reissig
et al. (1955), using N-acetyl D-glucosamine as the standard. One enzyme
unit was defined as the amount of enzyme that catalyzes the release of 1 µmol
of N-acetyl D-glucosamine in 1 hr at 45ºC.
The protein concentration was measured using the method of Lowry
et al. (1951) with bovine serum albumin as standard. For the purified
enzyme, protein concentration was measured by determining the absorbance
at 280 nm.
Purification of chitinase
The purification of chitinase was carried out in three steps.
The cell free supernatant was precipitated with 30% ammonium sulphate. The
resultant precipitate was centrifuged at 10,000 x g, 4ºC.
Then ammonium sulphate concentration was increased to 75% saturation and it
was left overnight at 4ºC. The precipitate was collected
by centrifugation at 10,000 x g, 4ºC.
It was dissolved in 50 ml of 25 mM Tris-HCl buffer pH 7.5 and dialysed
against the same buffer.
The dialysed protein was subjected to ion exchanger, DEAE-Sephadex
column (1.5 x 12 cm).
The adsorbed chitinase was eluted by a linear gradient of NaCl from 0 to 0.25 M in the same buffer. Chitinase
activity was assayed in each 5.0 ml fraction at a flow rate of 42 ml h-1.
In final step, the active fractions were pooled, concentrated by polyethylene
glycol and dialysed against Tris-HCl, pH 7.5 and loaded onto a gel filtration
column (2.2 cm x 90
cm), Sephadex G-200 and flow rate was maintained at 20
ml h-1. The molecular weight was estimated from a standard curve obtained from
the proteins with their molecular weights known (68 kDa, bovine serum albumin,
45 kDa, ovalalbumin and 30 kDa, casein).
The purified protein was loaded onto SDS-PAGE (12%) as described
by Laemmli (1970) to determine the protein profile. Native
PAGE was carried out with the aim to study the zymography pattern of chitinase.
The detailed procedure was exactly similar to the SDS-PAGE in which SDS, mercaptoethanol
and the heating step during protein sample preparation were eliminated. The
native PAGE gel was run with purified chitinase preparation. Half of the gel
was cut and stained to locate the position of single band and the other half
of the gel was placed over chitin agar plate (1.0% swollen chitin in citrate
phosphate buffer + 1.5% agar) and incubated overnight at 45ºC to find the zone of clearance.
Characterization of
purified chitinase
The purified chitinase was characterized with respect to its
optimum pH, temperature, stability at different temperatures and pH values,
effect of metal ions, surfactants, organic solvents on activity and stability.
Chitinase activity was assayed at different pH values (pH
2.6 to 10.0) using different buffers 50
mM such as citrate-phosphate buffer (pH, 2.6-7.0), sodium
phosphate buffer (pH, 6.5-8.0), tris-HCl buffer (pH, 7.0-8.5) and glycine-NaOH
buffer (pH, 8.6-10.0). To determine pH stability, chitinase preparations in
buffer at different pH ranging from 4.0-9.0 were kept at room temperature for
2 hrs. Thereafter chitinase activity was assayed under standard conditions.
Chitinase activity was assayed at different temperatures ranging
from 35-60ºC at pH 5.5 in citrate phosphate buffer (50
mM). To determine thermostability, chitinase preparation
was incubated at temperature ranging from 40-55ºC for
different time intervals up to 3 hrs. Chitinase activity was assayed at 45ºC and pH 5.5.
The effect of substrate concentration on chitinase activity
was determined at different concentrations of chitin, varying between 0.25
mg ml-1 to 16 mg ml-1 (w/v). The Km and Vmax values
were determined by Lineweaver-Burks plot.
The effect of metal ions on enzyme activity was studied by
incorporating these metal ions such as MgSO4. 7H2O, KCl,
CaCl2, 2H2O, CuCl2, 2H2O, HgCl2,
AgNO3, CoCl2, 2H2O, ZnSO4, FeCl3 and
FeSO4 in reaction mixture at 1
mM to100 mM concentration. Effects of these metal ions
on enzyme stability were studied by incubating the purified enzyme in 25 mM Tris-HCl buffer, pH 7.5, with these
metal ion salts at room temperature for 1 hr and subsequently determining the
residual enzyme activity under standard assay conditions.
Allosamidin was added to the enzyme solution in the concentration
range from 1 to 100 µg ml-1 and incubated at room temperature for
1 hr. Thereafter, residual enzyme activity was determined under standard assay
conditions. The effect of sugars such as N-acetyl D-glucosamine, glucosamine
HCl, galactosamine and glucose was studied by incorporating these sugars at 1 mM and 10 mM concentration in the reaction mixture
and subsequently determining the enzyme activity.
Substrate binding was determined by incubating the enzyme
with 10 mg substrate in citrate phosphate buffer (50
mM, pH 5.5) for 30 min at 0ºC with
intermittent shaking followed by centrifugation at 4ºC and
residual activity was determined in the supernatant under standard assay conditions.
Chemical modification of chitinase was done using several
reagents such as para chloromecuribenzoate (PCMB), N-bromosuccinimide (NBS),
5, 5'-dithiobis-(2-nitrobenzoic) acid (DTNB), iodoacetamide and methylene blue.
The effects of these modifiers were tested by incubating the enzyme with varying
concentrations (0.1 mM to 10 mM) of the modifiers in the reaction
mixture.
Hydrolysis pattern of
purified chitinase
The mode of action of chitinase was determined by viscometric
assay (Otakara, 1961). Purified chitinase (60 µg) was added
to 60 ml substrate solution (5g L-1 glycol chitin in 50
mM citrate phosphate buffer, pH, 5.5) and the mixture
was incubated at 45ºC for
digestion. Aliquots (10 ml each) were removed at intervals and subjected immediately
to viscosity measurement on an Ostwald viscometer. After incubation of the
enzyme with 1.0% swollen chitin at 45ºC for
5 hrs, the hydrolytic products of chitin were resolved by high performance
liquid chromatography (HPLC) (Shimadzu, USA).
The HPLC system was fitted with LC1oAT HPLC pumps and a SPD-M10A detector.
A reversed phase 100 NH2 (4 x 250
mm) column from Shimadzu was
used. The samples were eluted with 75% (v/v) acetonitrile in water with a flow
rate of 1.0 ml min-1, and the injection volume was 20 µl. The aliquots
were monitored for UV absorbance at 205 nm. N-acetyl D-glucosamine and chitobiose
were used as standards. The peak areas of standard solutions and hydrolyzates
were integrated by SPD-MXA real time software (Shimadzu, USA).
Results
Purification
of chitinase
With swollen chitin as the sole source of carbon, Enterobacter sp.
NRG4 produced chitinase in the culture medium. The chitinase was purified using
standard techniques i.e. ammonium sulphate precipitation (30-75%), DEAE-Sephadex
ion exchange chromatography and Sephadex G-200 gel filtration chromatography.
When cell free supernatant was subjected to fractional ammonium sulphate precipitation,
chitinase activity was precipitated in 30-75% salt saturation. The yield of
chitinase was 71% with a purification fold of 3.18 and specific activity of
560.5 U mg-1 protein. The dialyzed protein was loaded on DEAE ion
exchanger. After elution with 0 to 250
mM NaCl gradient two major peaks of proteins were observed
but chitinase activity was observed only in peak A. Here the yield of chitinase
was 47.1% with a purification fold of 23.2 and specific activity 4090.9 U mg-1.
Using gel filtration, the chitinase was purified by 44.12 fold with specific
activity of 7783.3 U mg-1 and the yield was 31.1%. The results of
chitinase purification are summarized in Table 1. The
molecular weight of the chitinase was estimated to be 60 kDa by SDS-PAGE (Figure
1a). It was consistent with the molecular mass determined by Sephadex G
200 gel filtration, suggesting that the purified chitinase is a monomer type.
Native gel electrophoresis showed a single band which corresponded to chitinase
activity as shown by the hydrolysis zone in the zymogram (Figure
1b).
Table 1. Purification of chitinase by ammonium
sulphate, DEAE-Sephadex column chromatography and Sephadex G-200 gel
filtration.
|
Purification Step
|
Total Activity
(U)
|
Total protein
(mg)
|
Specific activity
(U/mg)
|
Purification fold
|
Recovery
(%)
|
Cell free supernatant
|
45100
|
255
|
176.4
|
-
|
100
|
Dialysed (ammonium sulphate precipitation 30-75%)
|
31950
|
57
|
560.5
|
3.18
|
71.0
|
DEAE Sephadex
|
22500
|
5.5
|
4090.9
|
23.2
|
47.1
|
Gel filtration
(Sephadex G-200)
|
14010
|
1.8
|
7783.3
|
44.12
|
31.1
|
Characterization of
purified chitinase
The chitinase was maximally active at pH 4.5 to 8.0 thus exhibiting
a broad pH optima (Figure 2a). Determination of pH stability
of the chitinase indicated that the enzyme was stable between pH 4.5 to 8.0
and it retained 90% of its activity in this range (Figure
2b). The purified enzyme showed its maximum activity at 45ºC and
was stable at 40ºC for 3 hrs (Figure
3).
With acid swollen chitin, colloidal chitin, regenerated chitin
and glycol chitin the purified chitinase gave Km of 1.43 mg ml-1,
1.41 mg ml-1, 1.8 mg ml-1 and 2.0 mg ml-1,
respectively and Vmax were 83.33 µmole µg-1 h-1,
74.07 µmole µg-1 h-1, 40.00 µmole µg-1 h-1 and
33.33 µmole µg-1 h-1, respectively (Table
2).
Table
2. Kinetic parameters for chitin hydrolysis by Enterobacter sp.
NRG4 chitinase.
|
Substrate
(1.0%)
|
Km
(mg ml-1)
|
Vmax
(µmole µg-1 h-1)
|
Swollen chitin
|
1.43
|
83.33
|
Colloidal chitin
|
1.41
|
74.07
|
Regenerated chitin
|
1.8
|
40.00
|
Glycol chitin
|
2.0
|
33.33
|
The enzyme showed activities towards swollen chitin, colloidal
chitin, glycol chitin and regenerated chitin but exhibited no activity towards
carboxymethyl cellulose, chitosan and Micrococcus lysodeikticus cell
wall. When swollen chitin was used as substrate the activity was taken as 100.
The activities with colloidal chitin, regenerated chitins, glycol chitin, flake
chitin and crab shell chitin were 80.3, 44.7, 39.4, 5.9 and 2.3%, respectively. Enterobacter sp.
NRG4 chitinase reduced the viscosity of glycol chitin significantly in 5 min
due to cleavage of chitin long chains by the chitinase at 45ºC (Figure
4). Thus it was concluded that the purified chitinase has endo-splitting
activity. As shown in Figure 5 hydrolyzed products of
enzymatic reaction of purified enzyme were (GlcNAc)2 and N-acetyl
D-glucosamine.
Chitinase exhibited a substrate binding capacity of 89.5,
26.2 and 15.2% for swollen chitin, flake chitin and carboxymethyl cellulose,
respectively whereas no significant substrate binding was observed for pectin,
starch, xylan, wheat bran and chitosan (Figure 6).
Mg2+, K+ and Ca2+ stimulated
chitinase activity by 13, 16 and 18%, respectively whereas Cu2+,
Co2+, Ag+ and Hg2+ inhibited chitinase activity
by 9.7, 15, 22 and 72.2%, respectively at 1mM concentration. At 100
mM concentration Cu2+, Ag+ and
Hg2+ completely inhibited chitinase activity when incubated at room
temperature for 1 hr whereas Zn2+, Fe3+, Co2+ and
Fe2+ inhibited chitinase activity by 98.3, 90.0, 89.5 and 83.7%,
respectively.
Allosamidin, a known specific inhibitor of chitinase inhibited Enterobacter sp.
NRG4 chitinase by 57.1 and 65.7% at a concentration of 50 and 100 µg ml-1,
respectively, with an IC50 value of 40 µg ml-1 (64 µM)
(Figure 7). Study of end-products and sugars on chitinase
activity showed that N-acetyl D-glucosamine, glucosamine HCl, galactosamine
and glucose inhibited enzyme activity by 10, 8, 4 and 9.1% at 1
mM concentration and by 81.3, 19.0, 26.0 and 19.0%, respectively
at 10 mM concentration
of these sugars.
Iodoacetamide inhibited chitinase activity by 17.6, 66.2 and
84.5%, respectively at 0.1 mM, 1
mM and 5 mM concentration. DTNB inhibited chitinase
activity by 1.5, 30.6, 77.5 and 97.2%, respectively at 0.1 mM, 1 mM, 5 mM and 10 mM concentration, respectively. NBS
at 1 mM and iodoacetamide
at 10 mM concentration
completely inhibited the enzyme activity. PCMB did not affect the enzyme activity
significantly. EDTA at 1 mM concentration
inhibited chitinase activity by 11%.
Discussion
An extracellular chitinase secreted by Enterobacter sp.
NRG4 was purified to homogeneity by combination of ammonium sulphate precipitation,
DEAE Sephadex ion exchange chromatography and Sephadex G-200 gel flitration
chromatography. The chitinase showed a single band on 12% SDS-PAGE and Native
PAGE indicating the complete purification of the enzyme. The molecular weight
of the protein was found to be about 60 kDa by SDS-PAGE as well as by gel filtration
chromatography. The chitinase from Enterobacter sp. NRG4 was active
over broad pH range i.e. from pH 4.5-8.0, optimum being 5.5. Several
workers have reported broad pH optima like pH 4.5-7.5 of chitinase from Bacillus
cereus (Pleban et al. 1997), pH 5.0-8.0 for Aeromonas
hydrophila H-2330 (Hiraga et al. 1997), pH 7.5-9.0 for Bacillus sp.
BG-11 (Bhushan and Hoondal, 1998). The pH optima for other
chitinases reported were pH 4.0 for Aeromonas sp. No. 10S-24 (Ueda
et al. 1995), pH 5.0 for Alcaligenes xylosoxydans (Vaidya
et al. 2001) and Arthrobacter sp. NHB-10 (Okazaki
et al. 1999), pH 5.5 for Bacillus sp. WY22 (Woo and
Park, 2003), pH 6.0 for Enterobacter sp. G-1 (Park
et al. 1997), pH 5.4 and 6.6 for CHIT60 and CHIT100, respectively from Serratia
plymuthica HRO-C48 (Frankowski et al. 2001), pH 6.3 for Bacillus sp.
NCTU2 (Wen et al. 2002), pH 6.5 for Vibrio alginolyticus H-8
(Ohishi et al. 1996) and Vibrio sp. (Zhou
et al. 1999), pH 7.0 for Monascus purpureus (Wang et al. 2002),
pH 7.0-8.0 for Bacillus 13.26 (Yuli et al. 2004) and
pH 10.0 for Cellulomonas flavigena NTOU1 (Chen et al. 1997).
The chitinase from the present strain was stable over wide
pH range i.e. from pH 4.5 to 8.0. Other bacterial chitinase stable over
broad pH range were pH 4.0 to 9.0 of Aeromonas sp. No. 10S-24 chitinase
(Ueda et al. 1995), pH 6.0 to 9.0 of Pseudomonas aeruginosa K-187
(Wang and Chang, 1997), pH 5.0 to 8.0 of Aeromonas hydrophila H2330
chitinase (Hiraga et al. 1997), pH 4.0 to 9.0 for Vibrio sp.
(Zhou et al. 1999), pH 6.8 to 8.0 of Bacillus sp.
NCTU2 (Wen et al. 2002) chitinase and pH 4.0 to 8.5 of Bacillus
cereus strain 65 (Pleban et al. 1997).
The temperature activity and stability profile of Enterobacter sp.
NRG4 chitinase revealed that the enzyme was optimally active at 45ºC.
It was stable at 40ºC for
more than 3 hrs and for 1 hr at 45ºC.
It retained 84.4% activity after 3 hrs at 45ºC.
The temperature optima of Enterobacter sp. NRG4 was in accordance with
other reports in literature such as Arthrobacter sp. NHBN-10 (Okazaki
et al. 1999), Vibrio alginolyticus TK-22 (Ohishi et
al. 1996). Chitinase from Vibrio alginolyticus TK-22 was stable
at 40ºC for
30 min (Ohishi et al. 1996) and purified chitinase of Vibrio sp.
P-6-1 was stable at 40ºC but
completely inactivated at 55ºC in
30 min (Takahashi et al. 1993).
The Km values of the Enterobacter sp. NRG4
chitinase against different substrates were 1.43 mg ml-1, 1.41 mg
ml-1, 1.8 mg ml-1 and 2.0 mg ml-1, respectively
with swollen chitin, colloidal chitin, regenerated chitin and glycol chitin
respectively, which are comparatively lower than the other reports in literature.
The Km values of chitinase from different organisms were, 2.88 mg
ml-1 for Enterobacter aerogenes (Tang et al.
2001), 1.4 mg ml-1 and 0.8 mg ml-1 for chitinase
C1 and C3 from Vibrio alginolyticus H-8 against squid chitin (Ohishi
et al. 1996), 3.0 mg ml-1 for Alcaligenes xylosoxydans chitinase
(Vaidya et al. 2003) and Bacillus sp. WY22 chitinase
(Woo and Park, 2003), 12 mg ml-1 for Bacillus sp.
BG-11 chitinase (Bhushan and Hoondal, 1998).
Ethylene glycol chitin, glycol chitin and colloidal chitin
are useful substrate for enzyme assays of endo-type chitinase (Park
et al. 1997). The hydrolysis pattern of purified enzyme indicated that
chitinase from Enterobacter sp. NRG4 was an endochitinase. It exhibited
high activity towards swollen chitin, colloidal chitin, regenerated chitin
and glycol chitin as compared to flake chitin and crab shell chitin. It showed
no activity towards carboxymethyl cellulose, chitosan and Micrococcus lysodeikticus cell
wall. The hydrolysis products from swollen chitin were (GlcNAc)2 and
GlcNAc. Enterobacter sp. G-1 was also reported to secrete an endochitinase
which showed high activity towards colloidal chitin and ethylene glycol chitin
more than flake chitin or soluble CMC. It could not hydrolyze flake chitosan
but showed 36 to 80% activity towards deacetylated chitosan compared with colloidal
chitin. The products from colloidal chitin hydrolysis were mainly (GlcNAc)2 with
small amount of (GlcNAc)3 and (GlcNAc)4 (Park
et al. 1997). Characteristics of purified chitinases from other reported Enterobacter spp.
are summarized in Table 3. Aeromonas sp. chitinase
I and II hydrolyzed colloidal chitin and ethylene glycol chitin effectively
but the activity was significantly lower towards chitin and chitosan. No detectable
activities towards Micrococcus lysodeikticus cell wall were observed
(Ueda and Arai, 1992). Chitinase exhibited a substrate binding
capacity of 89.5, 26.2 and 15.2% for swollen chitin, flake chitin and carboxymethyl
cellulose, respectively. Lee et al. (2000) reported binding
of Pseudomonas sp. YHS-A2 chitinase 78, 12, 0, 5 and 10% with colloidal
chitin, chitin, carboxymethyl cellulose, crude chitosan and birch wood xylan,
respectively.
Table 3. Comparison of the characteristics of purified
chitinase from other reported Enterobacter sp.
|
Species
|
Chitinase
|
Mol. wt.
(kDa)
|
Optimum
pH
|
Optimum
temp. (ºC)
|
Substrate
|
Hydrolysis
product/s
|
Inhibitors
|
Reference
|
Enterobacter sp. NRG4
|
Endochitinase
|
60
|
5.5
|
45
|
Swollen chitin
|
(GlcNAc)(GlcNAc)2
|
Cu2+, Co2+, Ag+, Hg2+,
NBS, DTNB, iodoacetamide
|
Present study
|
Enterobacter sp. G-1
|
Endochitinase
|
60
|
7.0
|
40
|
Colloidal chitin
|
(GlcNAc)2
(GlcNAc)3 (GlcNAc)4
|
EDTA, PCMB
|
Park et al. 1992
|
Enterobacter aerogenes
|
N.D.
|
42.5
|
6.0
|
55
|
-
|
-
|
Hg2+, Co2+ Mg2+
|
Tang et al. 2001
|
Enterobacter agglomerans
|
Endochitinase
|
61
|
6.5
|
40
|
pNP-(GlcNAc)3
|
pNP
|
-
|
Chernin et al. 1997
|
Among metal ions, Mg2+, K+ and Ca2+ stimulated
chitinase activity by 13, 16 and 18%, respectively whereas Cu2+,
Co2+, Ag+ and Hg2+ and inhibited chitinase
activity by 9.7, 15, 22 and 72.2%, respectively at 1
mM concentration. Activation of chitinase by Ca2+ or
Mg2+ is rare and reported in few cases only. At 100 mM concentration Cu2+ and
Ag+ completely inhibited chitinase activity when incubated at room
temperature for 1 hr. In Pseudomonas aeruginosa, Mg2+ and
Na+ were inhibitory while Cu2+ activated the chitinase
by 50% (Wang and Chang, 1997). Stimulatory effect of Ca2+ (30%)
and Mn2+ (20%) at 1 mM concentration
on Pseudomonas sp. YHS-A2 chitinase has been reported by Lee
et al. (2000).
Serratia plymuthica activity was stimulated by 120,
150 and 240% in presence of 10 mM Ca2+,
Co2+ or Mn2+ and inhibited by 80% in presence of 10
mM Cu2+ (Frankowski et al. 2001).
Chitinase from Alcaligenes xylosoxydans was inhibited by 25% by Cu2+ and
Na+ at 5 mM but
not by Ca2+, Ba2+ or Mg2+ at the same concentration
(Vaidya et al. 2003). Enterobacter sp. G-1 chitinase
activity was not affected by addition of Ca2+ or NaCl to the enzyme
solution (Park et al. 1997). Chitinase from Enterobacter
aerogenes was stimulated by Zn2+, Ba2+, Ca2+ and
Mn2+ and strongly inhibited by Hg2+, Co2+ and
Mg2+ (Tang et al. 2001). Hg+ and Hg2+ inhibited
chitinase of Streptomyces sp. M-20 (Kim et al. 2003). Frankowski
et al. (2001) reported stimulation of CHIT60 from Serratia plymuthica HRO-C48
by 10 mM Ca2+, Co2+ or
Mn2+ and inhibition in presence of Cu2+. In contrast,
Mn2+ and Ca2+ inhibited chitinase of Bacillus sp.
13.26 (Yuli et al. 2004). Ag+ and Hg2+ inhibited
chitinase C1 and C3 from Vibrio alginolyticus H-8 (Ohishi
et al. 1996). Hg2+ also inhibited chitinases from Arthrobacter sp.
NHB-10 (Okazaki et al. 1999) and Aeromonas hydrophila H
2330 (Hiraga et al. 1997). Chitinase from Monascus purpureus CCRC31499
was stimulated by Fe2+ and strongly inhibited by Hg2+ (Wang
et al. 2002). Ag+ and Hg2+ inhibited chitinase from Ralstonia sp.
A-471 (Sutrisno et al. 2004). Enterobacter sp. NRG4
chitinase was inhibited by 11% in presence of 10 mM EDTA. EDTA at 10 mM concentration inhibited chitinase
of Enterobacter sp. G-1 by 42% (Park et al. 1997).
N-bromosuccinamide at 1mM and iodoacetamide at 10 mM concentration completely inhibited
the enzyme activity. PCMB does not affect the enzyme activity much. DTNB and
iodoacetamide inhibition suggested the role of cysteine residues in active
site. NBS is protein oxidizing agent. The oxidizing reaction is specific for
tryptophan and -SH groups and therefore suggested the role of tryptophan residues.
There are few reports in the literature on effect of group specific reagents
on chitinase activity. PCMB at 1 mM concentration inhibited chitinase
of Enterobacter sp. G-1 by 24% (Park et al. 1997).
PCMB was inhibitory for chitinase of Aeromonas sp. No. 10S-24 (Ueda
et al. 1995). Chitinase from Streptomyces sp. M-20 was completely
inhibited by PCMB (Kim et al. 2003). Chitinase of Pseudomonas sp.
YHS-A2 was inhibited by 90% in presence of N-bromosuccinimide at 1mM concentration
(Lee et al. 2000).
Allosamidin inhibited chitinase activity by 57.1 and 65.7%
at 50 and 100 µg ml-1, respectively. The IC50 value was
40µg ml-1 (64 µM). Other reported IC50 values were 48 µM
for Bacillus sp. BG-11 chitinase (Bhushan and Hoondal,
1999) and 9.0 µM for chitinase from human serum and leucocytes (Escott
and Adam, 1995).
Among various sugars and end products, chitinase was inhibited
by 81.3% in presence of N-acetyl D-glucosamine at 10mM concentration whereas
glucosamine HCl, galactosamine and glucose inhibited up to 19%. Chitinase of Metarhizium
anisopliae was inhibited by 28, 21 and 79% in presence of glucose, N-acetyl
D-glucosamine and D-glucosamine, respectively at 10
mM concentration (Pinto et al. 1997).
In conclusion, we have purified and characterized a chitinase
from newly isolated Enterobacter sp. NRG4. The capability of this chitinase
to hydrolyze chitin efficiently, lower end product inhibition, broad pH activity
and stability makes the enzyme industrially significant for biotechnological
applications, especially in production of chitobiose and N-acetyl D-glucosamine.
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