Singh et al.

for 16 h. The dried residues were then ground to powder form (40 mm mesh) and

stored in polythene bags at room temperature (30±2°C) till used as substrate for

alpha amylase production.

Preparation of Inoculum

Bacterial Inoculum was prepared by the method described by Gangadharan et al.,

(2006). A volume of 50 ml of nutrient broth was inoculated with a loopful of cells

from a 24h old slant and kept at 37°C in a rotary shaker (100 rpm). After 18 h of

incubation, 1 ml of this nutrient broth culture of Bacillus licheniformis was used

as the inoculum for solid state fermentation.

Solid state fermentation medium for production of Alpha amylase

Fermentation medium was prepared by the method described by Gangadharan

et al., (2006). Five grams of each of the dried substrate were placed in 250 ml

Erlenmeyer flasks and then moistened with mineral salt solution (K2HPO4, 2g/L;

NH4NO3, 10g/L; NaCl, 1g/L; MgCl2, 1g/L). Distilled water was added to the

mineral salt solution in order to maintain the moisture content of medium. The

fermentation media in the flasks were autoclaved at 121°C for 20 minutes and

cooled to about 30°C. The flasks were inoculated with 2% inoculum of Bacillus

licheniformis and the contents of the flask were mixed thoroughly to ensure

uniform distribution of the inoculum. The flasks were incubated at 37°C for 24 to

48 h in a shaking incubator operated at speed of 100 rpm.All the experiments were

run parallel in triplicates.

Extraction of alpha amylase and its estimation

After fermentation, the fermented matter in each flask was extracted by the

addition of different extraction medium like distilled water, 0.2M Phosphate

buffer (pH 7±0.2), 0.1% Tween-80 and Triton-X-100 to a total extract volume of

100 ml. The entire content was mixed thoroughly at 30°C for 1 h in rotary shaker

at 100 rpm and filtered using a Whatman filter paper no.1. The suspensions were

then centrifuged at 8000 rpm at 4°C for 10 minutes. The supernatant was carefully

collected and used as crude enzyme extract for the estimation of alpha amylase

activity.

Estimation of alpha amylase

The alpha amylase assay was carried out according to the method described by

Okolo et al., (1995). The reaction mixture consisted of 1.25 ml of 1 % soluble

starch, 0.5 ml of 0.2 M phosphate buffer (pH=7), and 0.25 ml of crude enzyme

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International Journal of Bioinformatics and Biological Sciences : v.2 n.3&4. Dec. 2014