A 60 kDa extracellular chitinase was purified to homogeneity Enterobacter sp. NRG4. The optimal temperature and pH for activity were 45ºC and pH 5.5, respectively. Mg²⁺, K⁺ and Ca²⁺ stimulated whereas Cu²⁺, Co ²⁺, Ag⁺ and Hg²⁺ inhibited chitinase activity. Purified chitinase hydrolyzed chitin in to chitobiose and N-acetyl D-glucosamine.

Chitin is composed of repeating N-acetyl D-glucosamine residues linked by β-1,4 bonds. 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. 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, India. N-acetyl D-glucosamine was obtained from Fluka and chitobiose from Sigma, Co. Enterobacter sp. NRG4 (Dahiya et al. 2005) was grown in chitin containing medium. The micro-organism was cultivated at 30ºC for 72 hrs with agitation at 150 rpm and chitinase activity was assayed in cell free supernatant by the method of Reissig et al. (1955). The protein concentration was measured using the method of Lowry et al. (1951).

The chitinase was purified using ammonium sulphate precipitation followed by anion exchange (DEAE-Sephadex) and gel filtration chromatography on Sephadex G-200. The molecular weight of the chitinase was determined by the method of Laemmli (1970). The optimum pH of chitinase was determined by incubating the enzymes in buffers with different pH values (pH 2.6 to 10.0) and effect of temperature was studied by incubating the reaction mixture at different temperatures. The effect of metal ions and sugars such as N-acetyl D-glucosamine, glucosamine HCl, galactosamine and glucose was studied by incorporating them in reaction mixture. Effect of allosamidin was studied by incubating it with the enzyme solution at room temperature for 1 hr. The effects of  chemical modifiers was tested by incubating them with the enzyme in the reaction mixture. The mode of action of chitinase was determined by viscometric assay (Otakara, 1961) and hydrolytic products were resolved by high performance liquid chromatography (HPLC) (Shimadzu, USA).

The chitinase was purified by 44.12 fold with specific activity of 7783.3 U mg^-1 and the yield was 31.1%. The molecular weight of the chitinase was estimated to be 60 kDa by SDS-PAGE. The chitinase was maximally active in pH range 4.5 to 8.0 (Figure 1). It was optimally active at 45ºC (Figure 2). Enterobacter sp. NRG4 chitinase has endo-splitting activity. The hydrolyzed products of chitin were (GlcNAc)₂and N-acetyl D-glucosamine. 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-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. Allosamidin at a concentration of 50 and 100 µg ml^-1 inhibited Enterobacter sp. NRG4 chitinase by 57.1 and 65.7%, respectively. Iodoacetamide, DTNB and NBS inhibited chitinase activity whereas PCMB did not affect the enzyme activity significantly.

An extracellular chitinase secreted by Enterobacter sp. NRG4 was purified to homogeneity. The molecular weight of the protein was found to be about 60 kDa. The pH and temperature optima were 5.5 and 45ºC, respectively. Chitin was hydrolyzed in an endo-splitting manner and end products were (GlcNAc)₂ and GlcNAc. Mg²⁺, K⁺ and Ca²⁺ stimulated chitinase activity whereas Cu²⁺, Co ²⁺, Ag⁺ and Hg²⁺ inhibited it. N-bromosuccinamide, DTNB and iodoacetamide inhibition suggested the role of cysteine residues in active site. Allosamidin inhibited chitinase activity by 57.1 and 65.7% at 50 and 100 µg ml^-1, respectively. The IC₅₀ value was 40 µg ml^-1 (64 µM). Among various sugars and end products, chitinase was inhibited by 81.3% in presence of N-acetyl D-glucosamine at 10 mM concentration whereas glucosamine HCl, galactosamine and glucose inhibited up to 19%. In conclusion, we have purified and characterized a chitinase from newly isolated Enterobacter sp. NRG4 which can be used for production of chitobiose and N-acetyl D-glucosamine.

DAHIYA, Neetu; TEWARI, Rupinder; TIWARI, Ram Prakash and HOONDAL, Gurinder Singh. Chitinase production in solid state fermentation by Enterobacter sp. NRG4 using statistical experimental design.Current Microbiology, 2005. In press.

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OTAKARA, A. Studies on the chitinolytic enzymes of black-kojimold. Part I.Viscometric determination of chitinase activity by application of glycol chitin as a new substrate. Agricultural Biological Chemistry, 1961, vol. 25, p. 50-54.

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