versão On-line ISSN 0717-3458
Electron. J. Biotechnol. v.8 n.2 Valparaíso ago. 2005
Chitinase from Enterobacter sp. NRG4: Its purification, characterization and reaction pattern
Ram P. Tiwari
Gurinder Singh Hoondal*
Keywords: chemical modification, chitinase, Enterobacter sp. NRG4, purification, substrate binding.
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
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 126.96.36.199) and exochitinases [(chitobiosidases and β-N-acetyl hexosaminidase (EC 188.8.131.52) ] 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.
Flake chitin was obtained from Hi-Media,
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
The assay mixture contained 1 ml swollen chitin and 0.5 ml enzyme solution. After incubation at
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.
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,
The dialysed protein was subjected to ion exchanger, DEAE-Sephadex column (1.5 x
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
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
Chitinase activity was assayed at different temperatures ranging from 35-
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
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
Substrate binding was determined by incubating the enzyme with 10 mg substrate in citrate phosphate buffer (
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 (
The mode of action of chitinase was determined by viscometric assay (Otakara, 1961). Purified chitinase (60 µg) was added to 60 ml substrate solution (
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
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
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).
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
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
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
Iodoacetamide inhibited chitinase activity by 17.6, 66.2 and 84.5%, respectively at
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
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.
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
Serratia plymuthica activity was stimulated by 120, 150 and 240% in presence of
N-bromosuccinamide at 1mM and iodoacetamide at
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
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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