Isolation, purification and characterization of phytase from Bacillus subtilis MJA

Abstract

In this study, three strains of bacteria were isolated from soil. Among the three isolated strains, one was identified morphologically and confirmed by the molecular techniques as Bacillus subtilis MJA with high phytase activity. The phytase-producing bacteria were isolated using phytate screening agar media (PSM) with only 1.5% glucose and 0.5% sodium phytate as only source for carbon. In order to optimize the phytase production by B. subtilis MJA, different factors were studied. A combination of 0.5% glucose and 0.5% sucrose showed to be the best carbon source. Also, malt extract used as a source of nitrogen gave the highest phytase production. Also, the maximum phytase production was detected after incubation for four days (720 U/ml) at an optimum pH value of 7. The produced phytase was purified through various chromatographic techniques. The estimated enzyme molecular mass was about 38 kDa and the phytase had an optimal temperature and pH of 37°C and 5 to 6, respectively. On the other hand, studying the enzyme stability showed that enzyme was stable at low temperature, and had good pH stability by retaining 80% of its initial activity over a wide range of pH from 2 to 8. Kinetic values of V_max and K_m for the purified enzyme were 510 U/mg and 0.485 mM, respectively. The phytase activity was affected by different divalent metal ions. Cations such as Cu²⁺ or Fe²⁺ showed an inhibition effect on the phytase activity and the effect was in a dose dependent manner while, cations such as Mg²⁺ or Ca²⁺ showed an increase in the phytase activity. On the other hand, among different matrices
used to immobilize the cells for phytase production, agar-agar matrix indicated a promising immobilization matrix used for phytase production by B. subtilis MJA.

Keywords: Phytase, microbial sources, optimization, purification, characterization, immobilization

African Journal of BiotechnologyVol. 12(20), pp. 2957-2967