Development of bioprocessing technique for the production and purification of phytate-degrading enzyme from Malaysian soil bacteria

Abstract

Due to several biological characteristics, bacterial phytate-degrading enzymes have considerable potential in commercial and environmental applications. Until now, there is no single phytase that is able to meet the diverse needs for all commercial and environmental applications. Phytase enzyme preparations have a wide range of applications in animal and human nutrition as well as bio fertilizer. Bacteria strains were isolated from Malaysian maize fields and roots, and screened for phytatedegrading enzyme activity. The production and purification of extra-cellular phytatedegrading enzyme from the most potential strain as phytase producer, Enterobacter sakazakiii ASUIA279 was optimized using response surface methodology (RSM) with full-factorial faced centered central composite design (FCCCD). An extracellular phytate-degrading enzyme synthesized by E. sakazakii ASUIA279 was purified to homogeneity using FPLC anion exchange chromatography and gel filtration. The arrangement of the bacterial isolates according to their ability to produce extra-cellular phytate-degrading enzymes were Enterobacter sakazakii ASUIA279 > Pantoea stewartii ASUIA271 > Bacillus cereus ASUIA260. The optimum combination of cultivation conditions for maximum phytate-degrading enzyme production were determined at incubation temperature which is 39.7 ºC, initial pH 7.1, rice bran percentage at 13.6 %, 320 rpm of agitation and 0 vvm of aeration. The optimum combinations of experimental conditions in ion exchange chromatography were at pH 5.1 and 47 mM of sodium acetate buffer. Its molecular mass was estimated to be 43 kDa by SDS-PAGE. The Machaelis constant (KM) and turnover number (kcat ) for sodium phytate at pH 5.0 and 50 °C were calculated from the Lineweaver-Burk plot to 760 µM and 4.14 s-1, respectively. Optimal activity was determined at pH 4.5 and 50 ± 5 °C. The enzyme was strongly inhibited by Fe3+, Cu2+, Zn2+, molybdate, vanadate, fluoride and phosphate (1 mM). In this study, an extracellular phytate-degrading enzyme with promising properties as an animal feed additives or soil nutrient enhancement produced by E. sakazakii ASUIA279, a newly isolated bacteria strain was obtained. The phytate-degrading enzyme synthesis by these bacteria was triggered by the high content of organic phytate in the rice bran. Rice bran could be utilized and developed as a media for the production of this enzyme.