Immobilization of Phytase Produced by Bacillus licheniformis

Abstract

The aim of present study was to prepare immobilized product for poultry industry. For this purpose phytase enzyme from previously isolated Bacillus licheniformis was immobilized on poultry feed bentonite. As phytase is of great importance in poultry because it degrades hazardous phytic acid so immobilization of phytase would allow us to make the enzyme cost-effective. First of all phytase production from Bacillus licheniformis using raw substrate wheat bran was optimized. Maximum production was obtained at pH 8, temperature SO°C and incubation period S days. After successful production enzyme was extracted by centrifugation. This extracted enzyme was precipitated with the help of acetone. As a result semi-purified enzyme was obtained and there was 4 folds increase in enzyme activity. Characterization of assay to quantitate phytase was carried out. Ideal conditions were obtained with 0.2 ml enzyme and 0.8 ml substrate and incubation time of 10 minutes. After assay characterization partially purified free enzyme, immobilization process and immobilized enzyme were characterized. The partially purified free enzyme showed maximum activity (88.S U/mg) at pH 8 and SO°C temperature, even at 60°C there was no significant decrease in activity. The effect of incubation time and pH was studied on immobilization process. There was no significant effect of incubation time was observed while immobilization yield was maximum at pH 8 (9S%) at pH 7 yield was (89%) while there was significant decrease in immobilization yield at pH S and pH 9. After immobilization optimization, effect of temperature and pH was studied on immobilized enzyme. This study revealed that immobilized enzyme was stable at higher temperature than free enzyme i.e. free enzyme showed maximum activity at SO°C and immobilized enzyme showed maximum activity at 70°C which is a beneficial character from industrial point of view. Moreover, only 14% activity was lost at pH 8 after immobilization. Desorption was carried out after adsorption. There was no significant loss of activity in wash 1, 2, 3 and 4. The loss in activity was 8.2 U/mg, 1.06U/mg, 2.S U/mg for wash 1, 2 and 3, respectively and there was no activity found in wash 4. Therefore, it was concluded that immobilization of thermostable phytase from Bacillus licheniformis on bentonite can be carried out successfully for application m poultry industry.