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Amylolytic activity and its parametric optimization of an endophytic bacterium Bacillus subtilis with an ethno-medicinal origin

Barman, Dina, Dkhar, Mamtaz Sahana
Biologia 2015 v.70 no.3 pp. 283-293
Bacillus subtilis, Dioscorea alata, EDTA (chelating agent), Potentilla siemersiana, bacteria, barium, biomass, calcium, endophytes, enzyme activity, enzymes, industrial applications, iron, isozymes, magnesium, manganese, metal ions, molecular weight, pH, polyacrylamide gel electrophoresis, sodium, specific growth rate, staining, starch, surfactants, temperature
Amylolytic enzymes are a starch hydrolyzing important group of industrial enzymes with a multi-dimensional utility. In the present study, endophytic bacteria were isolated from Potentilla fulgens Wall. and screened for their amylolytic activity. Out of total 13 endophytic bacteria isolated, 3 of them showed positive results for amylolytic activity, i.e. production of halo zone against blue background in starch-containing media. The most promising isolate was identified as Bacillus subtilis subsp. inaquosorum PR⁻¹. After 72 h of incubation, PR⁻¹ exhibited maximum biomass (0.017 mg) and the overall specific growth rate was recorded as 0.0126 h⁻¹. Amylolytic enzyme activity of PR⁻¹ was found to be optimal at 10 mg/mL starch concentration in pH 7 at 50°C temperature after 30 min of incubation. This indicates that the production of amylolytic enzyme by the representative isolate was growth-dependent as well as preferable for industrial application. The enzyme remained stable in the presence of surfactants, whereas it was inhibited by ethylenediaminetetraacetic acid. Metal ions Ca²⁺, Na⁺ and Fe²⁺ were found to increase the activity, while Ba²⁺, Mg²⁺ and Mn²⁺ inhibited the activity. Activity staining for amylolytic enzyme showing one major band in the starch native polyacrylamide gel electrophoresis confirmed its active amylolytic activity with absence of isozymes with a molecular weight of 97.4 kDa. The amylolytic activity of PR⁻¹ was also enumerated against the raw starch of Dioscorea alata L. where the highest amylolytic activity (50.46 U/mL) was achieved in 10 mg/mL of raw starch after 2 h of incubation.