STUDIES ON PRODUCTION AND CHARACTERIZATION OF MICROBIAL CHITINASE
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Date
2019-03
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UHF,NAUNI
Abstract
ABSTRACT
The present studies involved isolation, production and characterization of extracellular chitinase from
the rhizospheric soil samples of apple orchards of district Shimla and Kinnaur. In total, eighty five bacterial and
nine fungal isolates were screened for extracellular chitinase production. Among the thirty bacterial and three
fungal isolates producing extracellular chitinase, bacterial isolate SK1.2 and the fungal isolate SNF1.1 were
found to be the best. These two isolates were characterized morphologically and molecularly by using 16S and
18S rRNA gene sequencing technique. They were identified asBacillus cereus SK1.2|MF280165| and Alternaria
brassicicola SNF1.1|MK024281|. The culture conditions for extracellular chitinase production were standarized
for both the bacterial as well as fungal isolates with respect to temperature, pH, incubation time, carbon sources,
nitrogen sources and substrate concentration using One Variable at a Time (OVAT) approach. Maximum
chitinase production was observed at temperature 30˚C, pH 6.0 and 1% substrate concentration for both the
isolates, however incubation time of 72 h for bacterial isolate and 120 h for fungal isolate was found to be
optimum. Fructose and dextrose were found to be the best carbon sources for bacterial as well as fungal isolate
respectively whereas, ammonium chloride and yeast extract were found to be the best nitrogen sources in culture
broth. Two different substrates were tried (Rice bran and termite wings) for production of chitinase under Solid
state fermentation (SSF) and maximum production was found with rice bran. The enzyme from bacterial and
fungal isolates was partially purified to 3.10 and 3.15 folds respectively using ammonium sulfate precipitation
and gel filtration chromatography on Sephadex G-100 column. The partially purified enzymes showed single
band on Native-PAGE and single band with molecular weight of 43.7 and 44 kDa on SDS-PAGE for bacterial
and fungal isolate respectively. Enzymes were characterized and had a pH optima of 7.0 for bacterial chitinase
and 6.6 for fungal chitinase and optimum temperature of 40oC and 35oC for bacterial and fungal chitinase
respectively. Out of different metal ions (Cu2+, Mg2+, Ca2+, Fe2+, Mn2+ and Zn2+ ) tested for enzyme activity,
Mn2+ and Mg2+ were found to increase enzyme activity. Bacillus cereus SK1.2 chitinase enzyme showed
maximum activity at 0.5% of colloidal chitin, after which it falls slightly. The Vmax value was determined by
MM equation and confirmed by Lineweaver Burk plot. The value of Vmax and Km was 66.67 μmol/min and
0.13 mg/ml respectively. Bacterial chitinase showed stronger inhibitory activity towards potent phytopathogen
in comparsion of control i.e., 66.7% in case of Fusarium oxysporum, 64.6% in case of Rhizoctonia solani and
63% against Colletotrichum gloeosporioides. The fungi Fusarium oxysporum showed significant alterations in
its cell wall structure after treatment with purified chitinase as compared to control when observed under TEM
and STEM. Similarly, the partially purified chitinase enzyme from Alternaria brassicicola strain SNF1.1
showed strong inhibitory activity against Escherichia coli and Staphylococcus aureus. So, chitinase from these
organisms could be effectively employed as potential biocontrol agents against harmful pathogens
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